Antibodies that bind peptidoglycan recognition protein 1

ABSTRACT

Disclosed herein is a method for identifying TREM-1&#39;s ligand and antibodies, or fragments thereof, which are capable of modifying the function of TREM-1&#39;s ligand. Antibodies that reduce or block TREM-1 activation may be identified and selected using this method. Antibodies that bind to TREM-1&#39;s ligand and reduce TREM-1 activity may be suitable for use as medicaments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/483,390, filed Apr. 10, 2017, currently allowed; which is acontinuation of U.S. application Ser. No. 14/376,968, filed Aug. 6,2014, now U.S. Pat. No. 9,663,568; which is a 371 of InternationalApplication PCT/EP2012/074093, filed Nov. 30, 2012; which claims benefitof U.S. Provisional Application 61/598,968, filed Feb. 15, 2012, EP12158974.1, filed Mar. 12, 2012, and U.S. Provisional Application61/672,799, filed Jul. 18, 2012. The contents of all above-namedapplications are incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: 3338_0820005_ST25.txt; Size: 102,716 bytes; and Date ofCreation: Oct. 23, 2018) is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the field of immunology. Moreparticularly, the present invention relates to a method for theidentification of the ligand that is able to stimulate TriggeringReceptor Expressed on Myeloid cells (TREM-1), as well as antibodies thatbind TREM-1's ligand. Such antibodies are capable of modulating myeloidcell activation and, therefore, inflammatory responses.

BACKGROUND

Peptidoglycan recognition protein 1 (otherwise known as PGLYRP1, PGRP-S,TNFSF3L, PGRP, TAG7 and PGRPS) is expressed in neutrophils and releasedupon their activation. PGLYRP1 is highly abundant in diseased tissue andhas been shown to play an important role in the clearance of bacterialinfections by the innate immune system. The family of PGLYRP proteins(PGLYRP1, PGLYRP2, PGLYRP3, PGLYRP4) all interact with bacterialpeptidoglycans (PGNs) but there are important differences in theproteins' PGN binding sites. PGLYRP1 has an additional groovehypothesized to constitute a binding site for an unknown effector orsignalling protein (J. Mol. Biol. 347:683-691 (2005)). PGLYRP1 is ahighly conserved, 196 amino acid long protein consisting of a signalpeptide and a peptidoglycan binding domain.

The identification of a signalling mechanism mediated by PGLYRP1 isimportant in order to understand and thereby manipulate the function ofthis protein in various infectious and inflammatory diseases.

TREM-1, likewise, has well-described effects in immune modulation but,thus far, the mechanism leading to TREM-1 mediated immune function hasnot been understood. TREM-1 is a receptor expressed on myeloid cells,such as monocytes, macrophages and neutrophils. It is a transmembraneprotein consisting of 234 amino acids, including a single extracellularimmunoglobulin domain and a short cytoplasmic tail. TREM-1 has noapparent signalling motif but, when activated, forms dimers/multimersand mediates signalling by associating with the ITAM-containingsignalling adaptor protein, DAP12. Downstream signalling may includephosphorylation of Syk and Zap70. Downstream signalling may includeactivation of the NFAT, ELK, NK-kappaB transcription factor. When TREM-1is activated, it triggers the release of pro-inflammatory cytokines,such as TNF-α (TNF-alpha), IL-8 and monocyte chemotactic protein-1, frommyeloid cells.

TREM-1 is upregulated in patients with sepsis, rheumatoid arthritis (RA)and inflammatory bowel disease (IBD) and increasing evidence supportsthe theory that TREM-1 contributes to the development and progression ofinflammatory diseases. The blocking of TREM-1 signalling has furthermorebeen shown to have therapeutic activity in in vivo mouse models of RAand IBD.

The mode of action of TREM-1 activation has remained elusive because theligand that activates TREM-1 is not known in the art. Therefore, thereis a need in the art for a means of identifying TREM-1's ligand. Thereis a need in the art for a method of identifying a molecule, such as anantibody, that is capable of reducing, blocking, or interfering with theinteraction of TREM-1 with its ligand. There is a need in the art for amolecule, such as an antibody, that is capable of binding TREM-1'sligand and thus reducing, blocking, or interfering with the stimulationof TREM-1 by its ligand. There is a need in the art for a molecule, suchas an antibody, that is capable of binding TREM-1's ligand. There is aneed in the art for a molecule, such as an antibody, that is capable ofbinding TREM-1's ligand and thus blocking TREM-1 activation andsignalling. There is a need in the art for a molecule, such as anantibody, that is capable of binding TREM-1's ligand and thus reducingor blocking cytokine release from a myeloid cell expressing TREM-1.

Disclosed herein is a method and assay for identifying TREM-1's ligandand molecules, such as antibodies, that are capable of binding theligand of TREM-1. Described herein are antibodies that are capable ofinfluencing TREM-1 activation. Thus, the antibodies disclosed herein aresuitable for use as pharmaceuticals. Antibodies that bind the ligand ofTREM-1 and that reduce or block the interaction of TREM-1 with itsligand may have a substantial impact upon the quality of life ofindividuals with chronic inflammatory diseases such as rheumatoidarthritis, psoriatic arthritis and inflammatory bowel diseases.

SUMMARY

The invention relates to a method for identifying TREM-1's ligand andmolecules, such as antibodies, that bind to TREM-1's ligand, hereinidentified as being PGLYRP1. The invention also relates to PGLYRP1antibodies that may be identified by means of the invented method. Thus,the invention relates to PGLYRP1 antibodies that are capable ofmodifying the activation of TREM-1 by PGLYRP1, such as PGLYRP1antibodies that are capable of reducing TREM-1 activity (signallingand/or activation) by PGLYRP1. Antibodies that reduce TREM-1 activitymay be used for the treatment of inflammation.

The method for identifying a TREM-1 ligand comprises (a) culturing acell expressing TREM-1, a signalling protein for TREM-1 and a reporterconstruct that is activated by said signalling protein; (b) detecting,preferably quantifying, the activity of said cell expressing TREM-1 whenit is contacted with a cell, a compound or a fluid, such as a biologicalfluid or a tissue, that triggers TREM-1 activation; (c) contacting theculture of (b) with a TREM-1-activating component; (d) isolating thecomponent that binds TREM-1 and (e) characterising the isolatedcomponent. The ligand for TREM-1, identified by means of the currentinvention as being PGLYRP1, may be used to modify the activity ofTREM-1.

The method for identifying a molecule that specifically binds PGLYRP1and that modifies TREM-1 mediated cellular activity, comprises: (a)culturing the cell according to any one of embodiments 1-18; (b)detecting, preferably quantifying, the activity of said cell expressingTREM-1 when it is contacted with PGLYRP1 and, optionally, amultimerisation agent such as PGN; (c) contacting the culture of (b)with a molecule that specifically binds PGLYRP1; and (d) detecting,preferably quantifying, that the activity of said cell expressing TREM-1is less than or more than its activity as measured in (b).

The method for identifying a PGLYRP1 antibody that modifies TREM-1mediated cellular activity comprises: (a) culturing a cell expressingTREM-1, a signalling protein for TREM-1 and a reporter construct that isactivated by said signalling protein; (b) detecting, preferablyquantifying, the activity of said cell expressing TREM-1 when it iscontacted with PGLYRP1 and, optionally, in combination with amultimerising agent such as PGN; (c) contacting the culture of (b) withan antibody that binds PGLYRP1; and (d) detecting, preferablyquantifying, that the activity of said cell expressing TREM-1 is lessthan or more than its activity as measured in (b).

One method of identifying a PGLYRP1 antibody that decreases TREM-1mediated cellular activity comprises: (a) culturing a cell such as aT-cell expressing TREM-1, a signalling protein such as DAP12 and areporter gene such as luciferase or beta-galactosidase; (b) incubatingsaid cell with an activated neutrophil and, optionally, in combinationwith a multimerising agent such as PGN (c) detecting, preferablyquantifying, the luminescence of said cell; (d) contacting the cultureof the cell and the activated neutrophil with a PGLYRP1 antibody; and(e) detecting, preferably quantifying, that the luminescence of saidcell is less than the activity measured in (c).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that neutrophils activate the BWZ-TREM-1 reporter cellline. The BWZ reporter cell line expresses human TREM-1 and mediatesactivation of a NFAT-linked beta (β)-galactosidase reporter gene thatmay be quantitated by luminescence using the Beta-Glo Assay System kitfrom Promega, Denmark. The figure illustrates activation of the reportercell line when cultured with neutrophils in the presence of eithercytokine cocktails containing TNF-alpha (α), IL-6, IFN-gamma (γ) andGM-CSF or Toll-like Receptor (TLR) activating ‘cocktails’ (TLRL and TLR2mix (tlrl-kit2hm, Invivogen, Sigma-Aldrich, Denmark), as well as in thepresence of separate components of these activation mixtures.

FIGS. 2A-2D show the flow cytometric staining of PGN-stimulatedneutrophils with a TREM-1-tetramer recombinant protein. Control proteindoes not bind (FIG. 2A), whereas a

TREM-1-tetramer (SEQ ID NO: 2) binds to a subset of PGN-activatedneutrophils (FIG. 2B), and this can be competed by another TREM-1protein (FIG. 2D) but not by a control protein (FIG. 2C), confirming thespecificity of the interaction.

FIG. 3A and FIG. 3B: PGLYRP1 is identified by Immunoprecipitation(IP)/Mass spectroscopy (MS) with TREM-1. Soluble TREM-Fc was incubatedwith PGN-activated neutrophils, crosslinked, immunoprecipitated followedby cleanup, trypsin digestion and mass spectroscopy. Theimmunoprecipitation of the TREM-1 binding proteins resulted in 3specific proteins, 73 proteins overlap with the control protein, and 72were precipitated by the control protein alone (background (FIG. 3A)).The table (FIG. 3B) shows the results from the TREM-1 specificimmunoprecipitation and subsequent mass spectroscopy.

FIG. 4A and FIG. 4B show that soluble TREM-1 binds to PGLYRP1. Shownboth by flow cytometric staining with TREM-1 on HEK293 transfectantsexpressing recombinant PGLYRP1 (FIG. 4A) and by Biacore and ForteBioanalyses of the interaction between PGLYRP1 and a TREM-1 tetramer (SEQID NO: 2). Soluble human PGLYRP1 bound immobilized human TRENT-1 in thepresence and absence of 10 μg/ml soluble E. coli peptidoglycan (a PGN)(FIG. 4B). PGN by itself also bound immobilized human PGLYRP1 (FIG. 4C),and soluble human TREM-1 bound to immobilized PGLYRP1 before and afterthe PGLYRP1 surface was exposed to and bound by PGN (FIG. 4D).

FIG. 5A and FIG. 5B show activation of a TREM-1 reporter cell line byrecombinant PGLYRP1. Stimulation of a TREM-1 reporter cell line withrecombinant PGLYRP1 (cat. no. 2590-PG-050 R&D Systems Minneapolis Minn.,USA), as well as PGLYRP1 generated in-house (SEQ ID NO: 1) leads to adose dependent response in the presence of PGN (FIG. 5A). ThisPGLYRP1-induced response can be specifically blocked by TREM-1-Fc fusionprotein (FIG. 5B) validating a TREM-1 specific signal.

FIGS. 6A-6G show that monoclonal anti-PGLYRP1 antibodies can block theTREM-1 response in the reporter assay stimulated with PGN-activatedneutrophils. Examples of antibody hybridoma clone supernatants screenedin various plates for their ability to block the activation signal. Afew antibodies (those below the dotted line, such as F10, F95) are ableto block this signal. Black dots represent isotype control in each plate(FIG. 6A). FIG. 6B shows testing from another fusion given rise to 2additional blocking PGLYRP1 antibodies M-hPGRPS-2F5 and −2F7. Testing ofcommercially available anti-PGLYRP1 mAbs show that they are not able toblock this signal even at high doses, whereas the polyclonal,commercially available PGLYRP1 pAb (AF2590, R&D Systems MinneapolisMinn., USA) can. FIG. 6C shows a comparison of the commerciallyavailable anti-PGLYRP1 mAb 1880424 from Thermo Scientific (ThermoScientific, Waltham Mass., USA) with an isotype control (MAB002) and thepolyclonal anti-PGLYRP1 pAb (AF2590). FIG. 6D shows a comparison of thecommercially available anti-PGLYRP1 mAb 4H230 (US Biological, SalemMass., USA) with an isotype control (MAB002) and the polyclonalanti-PGLYRP1 pAb (AF2590). FIG. 6E shows a comparison of thecommercially available anti-PGLYRP1 mAb 9A319 (US Biological, SalemMass., USA) with an isotype control (MAB002) and the polyclonalanti-PGLYRP1 pAb (AF2590), FIG. 6F shows a comparison of thecommercially available anti-PGLYRP1 mAb 6D653 (Santa Cruz Biotechnology,Santa Cruz Calif., USA) with an isotype control (MAB002) and thepolyclonal anti-PGLYRP1 pAb (AF2590). The slight decrease in activityseen for the SC 6D653 mAb appears to be due to the azide-containingformulation since a PGLYRP1 independent signal triggering TREM-1 with 1μg/ml platebound anti-TREM-1 mAb shows the same phenomenon (FIG. 6G).

FIG. 7 shows that the TREM-1 ligand present in human RA synovial fluidis able to stimulate TREM-1.

FIG. 7 shows that plate-bound agonistic anti-TREM-1 mAb (R&D MAB1278,Minneapolis, Minn., USA) stimulates TREM-1 (star) and that an RAsynovial fluid (SF) sample to which PGN has been added displayed hTREM-1ligand activity which can be neutralized by a PGLYRP1 polyclonalantibody (AF2590), indicating that TREM-1 activity is PGLYRP1 dependent.

FIG. 8 shows the overall structure of the Type II PGLYPR1 construct. ICrepresents the intracellular domain, TM the transmembrane domain—both ofwhich originate from MDL-1 protein sequence, in combination with EC(extracellular) domain of hPGLYRP1.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 represents the amino acid sequence of mature, full lengthhPGLYRP1 peptide sequence.

SEQ ID NO: 2 represents a recombinant protein sequence containing thefollowing elements: human TREM-1 ECD, linker peptide, human TREM-1 ECD,human allotype IgG1 Fc with 7 mutations differing from wild type. (L15A,L16E, G18A, A111S, P112S, D137E, L139M) SEQ ID NO: 3 represents humanallotype IgG1 Fc with 5 mutations differing from wild type: L15A, L16E,G18A, A111S, P112S.

SEQ ID NO: 4 represents a recombinant protein sequence containing thefollowing elements from N to C terminus: 6xHIS tag, two copies of thestreptavidin binding protein domain (SBP), GS linker, C terminus ofcartilage oligomeric protein (COMP), GS linker, hDCIR-ECD.

SEQ ID NO: 5 represents a recombinant protein sequence containing thefollowing elements: hTREM-1-ECD GS linker, C terminus of cartilageoligomeric protein (COMP), GS linker, two copies of the streptavidinbinding protein domain (SBP), 6xHIS tag.

SEQ ID NO: 6 represents a recombinant protein sequence containing thefollowing elements in order from N to C terminus, human CD83 ECD, G4S×3linker peptide, human CD83 ECD, human allotype IgG1 Fc mutant.

SEQ ID NO: 7 represents full length human PGLYRP1 coding cDNA sequencewith C terminal GPI signal sequence. This sequence was cloned intopcDNA3.1zeo(+) (Invitrogen: V860-20, Carlsbad, Calif., USA) via EcoR1and Xho1 restriction sites.

SEQ ID NO: 8 represents mature full length hTREM-1 ECD (aa. 21-200).

SEQ ID NO: 9 represents cDNA for a CD33 leader tandem hTREMextracellular domains separated by a G4S×3 linker. A synthetic cDNA witha 5′ EcoRI restriction site, a GCCACC Kozak sequence the CD33 leadersequence followed by the extracellular domain of human TREM-1 (aa17-200)with a interspaced KpnI restriction site andglycine-glycine-glycine-serine spacer repeated three times (G4S×3)followed by an additional copy of the extracellular domain of humanTREM-1 (aa17-200) and an Apa1 site to allow cloning.

SEQ ID NO: 10 represents pentameric hTREM-COMP-SBP38x2-6HIS. EcoR1 siteand Kozak are 5′ and BamH1 site is 3′ of ORF.

SEQ ID NO: 11 represents hCD83 tetramer cloned into pJSV002-hFc6mutvector with EcoR1 and Apa1. EcoR1 site and Kozak are 5′ and Apa1 site is3′ of ORF.

SEQ ID NO: 12 represents pentameric 6HIS-SBP38x2-COMP-hDCIR. EcoR1 siteand Kozak 5′ and BamH1 3′ of ORF.

SEQ ID NO: 13 represents the nucleic acid sequence of the variable heavychain of a monoclonal PGLYRP1 antibody (1F36, mAb 0182).

SEQ ID NO: 14 represents the nucleic acid sequence of the variable lightchain of a monoclonal PGLYRP1 antibody (1F36, mAb 0182).

SEQ ID NO: 15 represents the amino acid sequence of the variable heavychain of a monoclonal PGLYRP1 antibody (1F36, mAb 0182).

SEQ ID NO: 16 represents the amino acid sequence of the variable lightchain of a monoclonal PGLYRP1 antibody (1 F36, mAb 0182).

SEQ ID NO: 17 represents the nucleic acid sequence of the variable heavychain of a monoclonal PGLYRP1 antibody (1 F10).

SEQ ID NO: 18 represents the nucleic acid sequence of the variable lightchain of a monoclonal PGLYRP1 antibody (1 F10).

SEQ ID NO: 19 represents the amino acid sequence of the variable heavychain of a monoclonal PGLYRP1 antibody (1F10).

SEQ ID NO: 20 represents the amino acid sequence of the variable lightchain of a monoclonal PGLYRP1 antibody (1F10).

SEQ ID NO: 21 represents the nucleic acid sequence of the variable heavychain of a monoclonal PGLYRP1 antibody (1F105, mAb 0184).

SEQ ID NO: 22 represents the nucleic acid sequence of the variable lightchain of a monoclonal PGLYRP1 antibody (1F105, mAb 0184).

SEQ ID NO: 23 represents the amino acid sequence of the variable heavychain of a monoclonal PGLYRP1 antibody (1F105, mAb 0184).

SEQ ID NO: 24 represents the amino acid sequence of the variable lightchain of a monoclonal PGLYRP1 antibody (1F105, mAb 0184).

SEQ ID NO: 25 represents the nucleic acid sequence of the variable heavychain of a monoclonal PGLYRP1 antibody (1F95).

SEQ ID NO: 26 represents the nucleic acid sequence of the variable lightchain of a monoclonal PGLYRP1 antibody (1F95).

SEQ ID NO: 27 represents the amino acid sequence of the variable heavychain of a monoclonal PGLYRP1 antibody (1F95).

SEQ ID NO: 28 represents the amino acid sequence of the variable lightchain of a monoclonal PGLYRP1 antibody (1F95).

SEQ ID NO: 29 represents the nucleic acid sequence of the variable heavychain of a monoclonal PGLYRP1 antibody (2F5).

SEQ ID NO: 30 represents the nucleic acid sequence of the variable lightchain of a monoclonal PGLYRP1 antibody (2F5).

SEQ ID NO: 31 represents the amino acid sequence of the variable heavychain of a monoclonal PGLYRP1 antibody (2F5).

SEQ ID NO: 32 represents the amino acid sequence of the variable lightchain of a monoclonal PGLYRP1 antibody (2F5).

SEQ ID NO: 33 represents the nucleic acid sequence of the variable heavychain of a monoclonal PGLYRP1 antibody (2F7).

SEQ ID NO: 34 represents the nucleic acid sequence of the variable lightchain of a monoclonal PGLYRP1 antibody (2F7).

SEQ ID NO: 35 represents the amino acid sequence of the variable heavychain of a monoclonal PGLYRP1 antibody (2F7).

SEQ ID NO: 36 represents the amino acid sequence of the variable lightchain of a monoclonal PGLYRP1 antibody (2F7).

SEQ ID NO: 37 represents the amino acid sequence of Type II 1.0 PGLYRP1.

SEQ ID NO: 38 represents the amino acid sequence of Type II 2.0 PGLYRP1.

SEQ ID NO: 39 represents the amino acid sequence of an epitope tag.

SEQ ID NO: 40 represents the amino acid sequence of full-length humanPGLYRP2.

SEQ ID NO: 41 represents the amino acid sequence of full-length humanPGLYRP3.

SEQ ID NO: 42 represents the amino acid sequence of full-length humanPGLYRP4.

SEQ ID NO: 43 represents the nucleic acid sequence of hCD33-hTrem1ECD(aa17-200)-Fc6mut.

SEQ ID NO: 44 represents the amino acid sequence of hCD33-hTrem1ECD(aa17-200)-Fc6mut.

SEQ ID NO: 45 represents the nucleic acid sequence for hCD33-hTremL1ECD(aa16-162)-Fc6mut.

SEQ ID NO: 46 represents the amino acid sequence of hCD33-hTremL1ECD(aa16-162)-Fc6mut.

SEQ ID NO: 47 represents the nucleic acid sequence for hCD33-hTremL2ECD(aa19-268)-Fc6mut.

SEQ ID NO: 48 represents the amino acid sequence of hCD33-hTremL2ECD(aa19-268)-Fc6mut

SEQ ID NO: 49 represents the nucleic acid sequence ofhCD33-hTREM2-Fc6mut dimer

SEQ ID NO: 50 represents the amino acid sequence of thehCD33-hTREM2-Fc6mut dimer.

SEQ ID NO: 51 represents the nucleic acid sequence of a primer.

SEQ ID NO: 52 represents the nucleic acid sequence of a primer.

SEQ ID NO: 53 represents the amino acid sequence of hCD33.

Description

The invention relates to a method for the identification of moleculessuch as antibodies that are capable of specifically binding TREM-1'ssignalling partner, herein identified as being PGLYRP1, and influencingPGLYRP1 binding with its signalling partner, TREM-1. PGLYRP1 may be usedto modify the activity of TREM-1. Hence, the invention relates tomolecules, such as antibodies, that influence inflammatory responsesthat are mediated by PGLYRP1. Antibodies that are capable of binding toPGLYRP1 and that influence TREM-1 activation and signalling have beencreated and identified.

A method or assay for the identification of TREM-1's ligand and foridentification of molecules, such as antibodies, that are capable ofspecifically binding PGLYRP1 and reducing or blocking PGLYRP1 activationof TREM-1 may be created as follows:

A first cell or population of first cells is transfected with genesencoding TREM-1 or fragments thereof, a signalling protein and areporter construct. The cell may be of haematopoietic origin, such as amyeloid cell, it may be a T-cell or it may be any other cell type thatis capable of being transfected and expressing such molecules. Thesignalling protein may be any protein that is capable of transmitting orconveying a signal, either directly or indirectly, from TREM-1 to thereporter construct and may include DAP10, DAP12, TCR zeta, Fc gammaRIII,an Fc receptor, or any other protein that is capable of transmitting orconveying a signal from TREM-1 to the reporter construct. Alternatively,the signalling protein may be a TREM-1/signalling chimera molecule. Thereporter construct comprises a transcription factor and a reporter gene,which in turn encodes a reporter protein that produces a detectablesignal, such as a quantifiable signal. The transcription factor may beNFAT or NFkB or any other suitable transcription factor known in theart. The reporter gene may encode beta (β)-galactosidase, luciferase,green fluorescent protein (GFP), chloramphenicol transferase or anyother reporter protein capable of producing a detectable signal. Onesuitable cell line that may be used for this bioassay is theBWZ.36/hTREM-1:DAP12:NFAT-LacZ T-cell (herein also identified as the“BWZ/hTREM-1 reporter cell”), the creation of which is described indetail in the examples. When activated, the BWZ/hTREM-1 reporter cellproduces beta (3)-galactosidase, the production of which may be measuredusing equipment or kits known in the art, such as Beta Glow™ (PromegaE4720, Madison, Wis., USA).

The first cell or population of first cells may be activated byincubation with PGLYRP1 and, optionally, a multimerisation agent. Theoptional multimerisation agent acts as a scaffold for PGLYRP1 and may bepeptidoglycan (PGN), neutrophil extracellular traps (NETs), hyaluronicacid, a proteoglycan structure such as versican, aggrecan, decorin orfibrin, or any other naturally occurring matrix structure or moleculethat is able to multimerise or present PGLYRP1. The first cell may beactivated by incubation with one or more second cell(s) thatexpressPGLYRP1 on its/their surface, or intracellularly. An example ofintracellular expression may be the storage of PGLYRP1 in secretorygranules. The second cell may thus be any cell (or population of cells)that expresses or is transfected with a gene encoding PGLYRP1 and thatexpresses PGLYRP1 on its surface. Such a second cell may be aprokaryotic or a eukaryotic cell, such as a mammalian cell, such as aCHO cell, a BHK cell or a HEK cell. The second cell may also be anactivated neutrophil. Neutrophils may be obtained from the whole bloodor tissue of an individual and used either in bulk or as purifiedneutrophils. Any agent that mimics the bacterial activation ofneutrophils, such as peptidoglycan (PGN) from the cell wall of abacterium, such as PGN-SA, PGN-EB, PGN-EC, PGN-BS (InVivogen,tlrl-pgnsa, San Diego, Calif.), may be used to activate a neutrophil.

The activity of the first cell or population of first cells is thendetected and, preferably, measured.

The culture of the first cell and the second cell(s) expressing PGLYRP1and/or the culture of the first cell incubated with PGLYRP1 and,optionally, a multimerisation agent such as PGN, is contacted with anantibody that has been raised against PGLYRP1. The activity of the firstcell or population of first cells is detected, and preferably measured.

In this way, antibodies that are capable of binding TREM-1's ligand,PGLYRP1, and that influence the interaction of PGLYRP1 with TREM-1 maybe identified, PGLYRP1 antibodies that cause an increase in the activityof the first cell enhance the interaction of PGLYRP1 with TREM-1 and areherein identified as “stimulating PGLYRP1 antibodies”. PGLYRP1antibodies that cause a decrease in the activity of the first cellreduce, interfere, or block the interaction of PGLYRP1 with TREM-1 andare herein identified as “inhibitory PGLYRP1 antibodies”. InhibitoryPGLYRP1 antibodies reduce or block TREM-1 activation and signalling.

Hence, the present invention relates to a method of characterising thefunction of PGLYRP1 antibodies. Antibodies capable of specificallybinding PGLYRP1 and that have any effect upon TREM-1 activation anddownstream signalling are herein referred to as “functional PGLYRP1antibodies”. Consequently, the term “functional PGLYRP1 antibodies” isintended to encompass both stimulating PGLYRP1 antibodies and inhibitoryPGLYRP1 antibodies.

Furthermore, the present invention relates to antibodies that arecapable of specifically binding PGLYRP1 and reducing, interfering with,or blocking its interaction with TREM-1, hence reducing TREM-1activation and downstream signalling. Antibodies of the invention mayhave an immunoregulatory function, reducing the cytokine production ofmyeloid cells expressing TREM-1. For example, antibodies of theinvention may reduce or prevent release of TNF-alpha (a), IL-1beta (b),IL-6, IFN-gamma (γ), MIP-1beta (b), MCP-1, IL-8 and/or GM-CSF frommyeloid cells such as macrophages and/or neutrophils and/or myeloidcells in diseased tissue such as synovial tissue. Antibodies of theinvention may be capable of down-regulating neutrophil responses.

PGLYRP1 antibodies according to the invention may reduce or block TREM-1activation by means of one or a combination of several differentmechanisms, affecting TREM-1 directly or indirectly. Antibodies of theinvention may prevent PGLYRP1 from creating a functional complex withTREM-1.

Antibodies of the invention may block PGLYRP1 function by reducing orblocking TREM-1 activation and downstream signalling.

The present invention also relates to inhibitory PGLYRP1 antibodies thatmay be identified by other means than the method disclosed herein.

Antibodies of the invention may be capable of binding both human PGLYRP1and PGLYRP1 from a species other than a human being. The term “PGLYRP1”,as used herein, thus encompasses any naturally occurring form of PGLYRP1which may be derived from any suitable organism, such as an invertebratespecies or a vertebrate species. PGLYRP1 for use as described herein maybe vertebrate PGLYRP1, such as mammalian PGLYRP1, such as PGLYRP1 from aprimate (such as a human, a chimpanzee, a cynomolgus monkey or a rhesusmonkey); a rodent (such as a mouse or a rat), a lagomorph (such as arabbit), or an artiodactyl (such a cow, sheep, pig or camel), amongothers. Preferably, the PGLYRP1 is human PGLYRP1 (SEQ ID NO: 1). ThePGLYRP1 may be a mature form of PGLYRP1 such as a PGLYRP1 protein thathas undergone post-translational processing within a suitable cell. Sucha mature PGLYRP1 protein may, for example, be glycosylated. The PGLYRP1may be a full length PGLYRP1 protein. The PGLYRP1 may be a splicevariant.

Antibodies of the invention may also be capable of specifically bindingvariants of PGLYRP1 such as SEQ ID NO: 37 (Type II 1.0 PGLYRP1) and/orSEQ ID NO: 38 (Type II 1.0 PGLYRP1).

Antibodies of the invention may be capable of influencing, such asinhibiting/reducing/blocking, the activity (signalling and/oractivation) of both human TREM-1 and TREM-1 from another species than ahuman being. The term “TREM-1”, as used herein, thus encompasses anynaturally occurring form of TREM-1 which may be derived from anysuitable organism. For example, TREM-1 for use as described herein maybe vertebrate TREM-1, such as mammalian TREM-1, such as TREM-1 from aprimate (such as a human, a chimpanzee, a cynomolgous monkey or a rhesusmonkey); a rodent (such as a mouse or a rat), a lagomorph (such as arabbit), or an artiodactyl (such a cow, sheep, pig or camel), amongothers. Preferably, the TREM-1 is human TREM-1. The TREM-1 may be amature form of TREM-1 such as a TREM-1 protein that has undergonepost-translational processing within a suitable cell. Such a matureTREM-1 protein may, for example, be glycosylated. The TREM-1 may be afull length TREM-1 protein. The TREM-1 may be a splice variant.

The term “antibody” herein refers to a protein, derived from a germlineimmunoglobulin sequence, which is capable of specifically binding toPGLYRP1 or a portion thereof. The term includes full length antibodiesof any isotype (that is, IgA, IgE, IgG, IgM and/or IgY) and any singlechain or fragment thereof. An antibody that specifically binds toPGLYRP1, or portion thereof, may bind exclusively to PGLYRP1, or portionthereof, or it may bind to a limited number of homologous antigens, orportions thereof.

Antibodies of the invention may be monoclonal antibodies, in the sensethat they may be directly or indirectly derived from a single clone of aB lymphocyte. An antibody of the invention may be a monoclonal antibody,with the proviso that it is not 188C424 (Thermo Scientific), 4H230 or9A319 (US Biological) or Clone 6D653 (Santa Cruz Biotechnology).

Antibodies of the current invention may be isolated. The term “isolatedantibody” refers to an antibody that has been separated and/or recoveredfrom another/other component(s) of its natural environment and/orpurified from a mixture of components in its natural environment.

Antibodies may be recombinantly expressed in prokaryotic cells,eukaryotic cells or an acellular system derived from cellular extracts.The prokaryotic cell may be E. coli. The eukaryotic cell may be a yeast,insect or mammalian cell, such as a cell derived from an organism thatis a primate (such as a human, a chimpanzee, a cynomolgus monkey or arhesus monkey), a rodent (such as a mouse or a rat), a lagomorph (suchas a rabbit) or an artiodactyl (such a cow, sheep, pig or camel).Suitable mammalian cell lines include, but are not limited to, HEK293cells, CHO cells and HELA cells. PGLYRP1 antibodies may also be producedby means of other methods known to the person skilled in the art, suchas a phage display or a yeast display. Antibodies of the invention maybe raised in vivo by immunising a suitable mammal with PGLYRP1, a cellexpressing PGLYRP1 or a combination of both.

PGLYRP1 antibodies may be produced, screened and purified using, forexample, the methods described in the Examples. In brief, any suitablemouse, including a PGLYRP1 knock-out (KO) mouse or a TREM-1 KO mouse,may be immunised with PGLYRP1, a cell expressing PGLYRP1 or acombination of both. Primary screening of hybridoma supernatants may beperformed using direct ELISA or FMAT and secondary screening may beperformed using flow cytometry. Positive hybridoma supernatants, as wellas purified antibodies, may then be screened for binding to, forexample, full length PGLYRP1. Positive hybridoma supernatants orpurified antibodies may then be tested for their ability to reduce orblock PGLYRP1-stimulation of TREM-1-bearing cells. The method of thecurrent invention may be used for this purpose.

Full-length antibodies of the invention may comprise at least fourpolypeptide chains: that is, two heavy (H) chains and two light (L)chains that are interconnected by disulfide bonds. One immunoglobulinsub-class of particular pharmaceutical interest is the IgG family, whichmay be sub-divided into isotypes IgG1, IgG2, IgG3 and IgG4. IgGmolecules are composed of two heavy chains, interlinked by two or moredisulfide bonds, and two light chains, each attached to a heavy chain bya disulfide bond. A heavy chain may comprise a heavy chain variableregion (VH) and up to three heavy chain constant (CH) regions: CH1, CH2and CH3. A light chain may comprise a light chain variable region (VL)and a light chain constant region (CL). VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). VH and VL regions aretypically composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The hypervariable regions of the heavy andlight chains form a [binding] domain that is capable of interacting withan antigen (PGLYRP1), whilst the constant region of an antibody maymediate binding of the immunoglobulin to host tissues or factors,including but not limited to various cells of the immune system(effector cells), Fc receptors and the first component (Clq) of theclassical complement system.

Examples of antigen-binding fragments include Fab, Fab′, F(ab)₂,F(ab′)₂, F(ab)S, Fv (typically the VL and VH domains of a single arm ofan antibody), single-chain Fv (scFv; see e.g. Bird et al., Science 1988;242:42S-426; and Huston et al. PNAS 1988; 85:5879-5883), dsFv, Fd(typically the VH and CHI domain), and dAb (typically a VH domain)fragments; VH, VL, VhH, and V-NAR domains; monovalent moleculescomprising a single VH and a single VL chain; minibodies, diabodies,triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al. ProteinEng 1997; 10: 949-57); camel IgG; IgNAR; as well as one or more isolatedCDRs or a functional paratope, where the isolated CDRs orantigen-binding residues or polypeptides can be associated or linkedtogether so as to form a functional antibody fragment. Various types ofantibody fragments have been described or reviewed in, e.g., Holligerand Hudson, Nat Biotechnol 2005; 2S: 1126-1136; WO2005040219, andpublished U.S. Patent Applications 20050238646 and 20020161201.

Certain antigen-binding fragments of antibodies may be suitable in thecontext of the current invention, as it has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. The term “antigen-binding fragment” of anantibody refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen, such as human PGLYRP1 orPGLYRP1 from another species, as described herein. Examples ofantigen-binding fragments include Fab, Fab′, F(ab)₂, F(ab′)₂, F(ab)S, Fv(typically the VL and VH domains of a single arm of an antibody),single-chain Fv (scFv; see e.g. Bird et al., Science 1988; 242:42S-426;and Huston et al. PNAS 1988; 85:5879-5883), dsFv, Fd (typically the VHand CHI domain), and dAb (typically a VH domain) fragments; VH, VL, VhH,and V-NAR domains; monovalent molecules comprising a single VH and asingle VL chain; minibodies, diabodies, triabodies, tetrabodies, andkappa bodies (see, e.g., Ill et al. Protein Eng 1997;10:949-57); camelIgG; IgNAR; as well as one or more isolated CDRs or a functionalparatope, where the isolated CDRs or antigen-binding residues orpolypeptides can be associated or linked together so as to form afunctional antibody fragment. Various types of antibody fragments havebeen described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol2005; 2S:1126-1136; WO2005040219, and published U.S. Patent Applications20050238646 and 20020161201. These antibody fragments may be obtainedusing conventional techniques known to those of skill in the art, andthe fragments may be screened for utility in the same manner as intactantibodies.

An antibody of the invention may be a human antibody or a humanisedantibody. The term “human antibody”, as used herein, is intended toinclude antibodies having variable regions in which at least a portionof a framework region and/or at least a portion of a CDR region arederived from human germline immunoglobulin sequences. (For example, ahuman antibody may have variable regions in which both the framework andCDR regions are derived from human germline immunoglobulin sequences.)Furthermore, if the antibody contains a constant region, the constantregion is also derived from human germline immunoglobulin sequences. Thehuman antibodies of the invention may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo).

Such a human antibody may be a human monoclonal antibody. Such a humanmonoclonal antibody may be produced by a hybridoma which includes a Bcell obtained from a transgenic nonhuman animal, e.g., a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene fused to an immortalized cell.

Human antibodies may be isolated from sequence libraries built onselections of human germline sequences, further diversified with naturaland synthetic sequence diversity.

Human antibodies may be prepared by in vitro immunisation of humanlymphocytes followed by transformation of the lymphocytes withEpstein-Barr virus.

The term “human antibody derivative” refers to any modified form of thehuman antibody, such as a conjugate of the antibody and another agent orantibody.

The term “humanised antibody”, as used herein, refers to ahuman/non-human chimeric antibody that contains one or more sequences(CDR regions) derived from a non-human immunoglobulin. A humanisedantibody is, thus, a human immunoglobulin (recipient antibody) in whichat least residues from a hypervariable region of the recipient arereplaced by residues from a hypervariable region of a non-human species(donor antibody) such as from a mouse, rat, rabbit, or non-humanprimate, which have the desired specificity, affinity, and capacity. Insome instances, FR residues of the human immunoglobulin are replaced bycorresponding non-human residues. An example of such a modification isthe introduction of one or more so-called back-mutations.

Furthermore, humanised antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, a humanised antibody will comprise at least one—typicallytwo—variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and in whichall or substantially all of the FR residues are those of a humanimmunoglobulin sequence. The humanised antibody can, optionally, alsocomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin.

The term “humanised antibody derivative” refers to any modified form ofthe humanised antibody, such as a conjugate of the antibody and anotheragent or antibody.

The term “chimeric antibody”, as used herein, refers to an antibodywhose light and heavy chain genes have been constructed, typically bygenetic engineering, from immunoglobulin variable and constant regiongenes that originate from different species. For example, the variablesegments of genes from a mouse monoclonal antibody may be joined tohuman constant segments.

The fragment crystallisable region (“Fc region”/“Fc domain”) of anantibody is the N-terminal region of an antibody, which comprises theconstant CH2 and CH3 domains. The Fc domain may interact with cellsurface receptors called Fc receptors, as well as some proteins of thecomplement system. The Fc region enables antibodies to interact with theimmune system. In one aspect of the invention, antibodies may beengineered to include modifications within the Fc region, typically toalter one or more of its functional properties, such as serum half-life,complement fixation, Fc-receptor binding, protein stability and/orantigen-dependent cellular cytotoxicity, or lack thereof, among others.Furthermore, an antibody of the invention may be chemically modified(e.g., one or more chemical moieties can be attached to the antibody) orbe modified to alter its glycosylation, again to alter one or morefunctional properties of the antibody. Preferably, a modified Fc domaincomprises one or more, and perhaps all of the following mutations thatwill result in decreased affinity to certain Fc receptors (L234A, L235E,and G237A) and in reduced C1q-mediated complement fixation (A3305 andP331S), respectively (residue numbering according to the EU index).

The isotype of an antibody of the invention may be IgG, such as IgG1,such as IgG2, such as IgG4. If desired, the class of an antibody may be“switched” by known techniques. For example, an antibody that wasoriginally produced as an IgM molecule may be class switched to an IgGantibody. Class switching techniques also may be used to convert one IgGsubclass to another, for example: from IgG1 to IgG2 or IgG4; from IgG2to IgG1 or IgG4; or from IgG4 to IgG1 or IgG2. Engineering of antibodiesto generate constant region chimeric molecules, by combination ofregions from different IgG subclasses, can also be performed.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further for instancein U.S. Pat. No. 5,677,425 by Bodmer et al.

The constant region may further be modified to stabilise the antibody,e.g., to reduce the risk of a bivalent antibody separating into twomonovalent VH-VL fragments. For example, in an IgG4 constant region,residue S241 may be mutated to a proline (P) residue to allow completedisulphide bridge formation at the hinge (see, e.g., Angal et al.,Mollmmunol. 199S; 30:105-8).

Antibodies or fragments thereof may also be defined in terms of theircomplementarity-determining regions (CDRs). The term“complementarity-determining region” or “hypervariable region”, whenused herein, refers to the regions of an antibody in which amino acidresidues involved in antigen binding are situated. The CDRs aregenerally comprised of amino acid residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light-chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy-chain variable domain; (Kabat et al. (1991)

Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242)and/or those residues from a “hypervariable loop” (residues 26-32 (L1),50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32(H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain;Chothia and Lesk, J. Mol. Biol 1987; 196:901-917). Typically, thenumbering of amino acid residues in this region is performed by themethod described in Kabat et al., supra. Phrases such as “Kabatposition”, “Kabat residue”, and “according to Kabat” herein refer tothis numbering system for heavy chain variable domains or light chainvariable domains. Using the Kabat numbering system, the actual linearamino acid sequence of a peptide may contain fewer or additional aminoacids corresponding to a shortening of, or insertion into, a framework(FR) or CDR of the variable domain. For example, a heavy chain variabledomain may include amino acid insertions (residue 52a, 52b and 52caccording to Kabat) after residue 52 of CDR H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc., according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

The term “framework region” or “FR” residues refer to those VH or VLamino acid residues that are not within the CDRs, as defined herein.

An antibody of the invention may comprise a CDR region from one or moreof the specific antibodies disclosed herein, such as a CDR region fromwithin SEQ ID NOs: 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35 or 36.

The 1F36 antibody has a heavy chain as shown in SEQ ID NO: 15 and alight chain as shown in SEQ ID NO: 16. An antibody of the invention maycomprise this variable heavy chain sequence and/or this variable lightchain sequence. The 1F36 antibody has the CDR sequences shown at aminoacids 31 to 35, 50 to 66 and 98 to 108 of SEQ ID NO: 15 and amino acids24 to 34, 51 to 56 and 89 to 97 of SEQ ID NO: 16. An antibody of theinvention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

An antibody according to the invention may comprise: a CDRH1 sequencethat corresponds to amino acid residues 31 to 35 (SWAM) of SEQ ID NO:15, wherein one of these amino acid residues may be substituted by adifferent amino acid residue; and/or a CDRH2 sequence that correspondsto amino acids 50 to 66 (MIHPSDSETRLNQKFKD) of SEQ ID NO: 15, whereinone, two or three of these amino acids may be substituted by a differentamino acid residue; and/or a CDRH3 sequence that corresponds to aminoacid residues 98 to 108 (DYSDYDGFAY]) of SEQ ID NO: 15, wherein one, twoor three of these amino acid residues may be substituted by a differentamino acid.

An antibody according to the invention may comprise: a CDRL1 sequencethat corresponds to amino acid residues 24 to 34 (RASQSISDYLH) of SEQ IDNO: 16, wherein one, two or three of these amino acid residues may besubstituted with a different amino acid; and/or a CDRL2 sequence thatcorresponds to amino acid residues 51 to 56 (ASQSIS) of SEQ ID NO: 16,wherein one or two of these amino acid residues may be substituted witha different amino acid; and/or a CDRL3 sequence that corresponds toamino acid residues 89 to 97 (QNGHSFPLT) of SEQ ID NO: 16, wherein oneor two of these amino acid residues may be substituted with a differentamino acid.

The 1F10 antibody has a heavy chain as shown in SEQ ID NO: 19 and alight chain as shown in SEQ ID NO: 20. An antibody of the invention maycomprise this variable heavy chain sequence and/or this variable lightchain sequence. The 1F10 antibody has the CDR sequences shown at aminoacids 31 to 35, 50 to 66 and 99 to 109 of SEQ ID NO: 19 and amino acids24 to 33, 49 to 55 and 88 to 96 of SEQ ID NO: 20. An antibody of theinvention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

An antibody according to the invention may comprise: a CDRH1 sequencethat corresponds to amino acid residues 31 to 35 (DYNMY) of SEQ ID NO:19, wherein one of these amino acid residues may be substituted by adifferent amino acid residue; and/or a CDRH2 sequence that correspondsto amino acids 50 to 66 (YIDPYNGDTSYNQKFKG) of SEQ ID NO: 19, whereinone, two or three of these amino acids may be substituted by a differentamino acid residue; and/or a CDRH3 sequence that corresponds to aminoacid residues 99 to 109 (GDYGNPFYLDY) of SEQ ID NO: 19, wherein one, twoor three of these amino acid residues may be substituted by a differentamino acid.

An antibody according to the invention may comprise a CDRL1 sequencethat corresponds to amino acid residues 24 to 33 (SVSSSVNYMY) of SEQ IDNO: 20, wherein one, two or three of these amino acid residues may besubstituted with a different amino acid; and/or a CDRL2 sequence thatcorresponds to amino acid residues 49 to 55 (DTSKLPS) of SEQ ID NO: 20,wherein one or two of these amino acid residues may be substituted witha different amino acid; and/or a CDRL3 sequence that corresponds toamino acid residues 88 to 96 (QQWTSNPPT) of SEQ ID NO: 20, wherein oneor two of these amino acid residues may be substituted with a differentamino acid.

The 1F105 antibody has a heavy chain as shown in SEQ ID NO: 23 and alight chain as shown in SEQ ID NO: 24. An antibody of the invention maycomprise this variable heavy chain sequence and/or this variable lightchain sequence. The 1F105 antibody has the CDR sequences shown at aminoacids 31 to 35, 50 to 66 and 99 to 108 of SEQ ID NO: 23 and amino acids24 to 33, 49 to 55 and 88 to 96 of SEQ ID NO: 24. An antibody of theinvention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

An antibody according to the invention may comprise: a CDRH1 sequencethat corresponds to amino acid residues 31 to 35 (DTYIH) of SEQ ID NO:23, wherein one of these amino acid residues may be substituted by adifferent amino acid residue; and/or a CDRH2 sequence that correspondsto amino acids 50 to 66 (RIDPANDDTKYDPNFQG) of SEQ ID NO: 23, whereinone, two or three of these amino acids may be substituted by a differentamino acid residue; and/or a CDRH3 sequence that corresponds to aminoacid residues 99 to 108 (SDNSDSWFAY) of SEQ ID NO: 23, wherein one, twoor three of these amino acid residues may be substituted by a differentamino acid.

An antibody according to the invention may comprise: a CDRL1 sequencethat corresponds to amino acid residues 24 to 33 (SVSSSVNFMN) of SEQ IDNO: 24, wherein one, two or three of these amino acid residues may besubstituted with a different amino acid; and/or a CDRL2 sequence thatcorresponds to amino acid residues 49 to 55 (DTSKLAP) of SEQ ID NO: 24,wherein one or two of these amino acid residues may be substituted witha different amino acid; and/or a CDRL3 sequence that corresponds toamino acid residues 88 to 96 (HQWSSYSLT) of SEQ ID NO: 24, wherein oneor two of these amino acid residues may be substituted with a differentamino acid.

The 1F95 antibody has a heavy chain as shown in SEQ ID NO: 27 and alight chain as shown in SEQ ID NO: 28. An antibody of the invention maycomprise this variable heavy chain sequence and/or this variable lightchain sequence. The 1F95 antibody has the CDR sequences shown at aminoacids 31 to 35, 50 to 66 and 99 to 106 of SEQ ID NO: 27 and amino acids24 to 33, 49 to 54 and 87 to 95 of SEQ ID NO: 28. An antibody of theinvention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

An antibody according to the invention may comprise: a CDRH1 sequencethat corresponds to amino acid residues 31 to 35 (DYNMH) of SEQ ID NO:27, wherein one of these amino acid residues may be substituted by adifferent amino acid residue; and/or a CDRH2 sequence that correspondsto amino acids 50 to 66 (YVDPYDGGTSSNQKFKG) of SEQ ID NO: 27, whereinone, two or three of these amino acids may be substituted by a differentamino acid residue; and/or a CDRH3 sequence that corresponds to aminoacid residues 99 to 106 (EVPYYFDY) of SEQ ID NO: 27, wherein one, two orthree of these amino acid residues may be substituted by a differentamino acid.

An antibody according to the invention may comprise: a CDRL1 sequencethat corresponds to amino acid residues 24 to 33 (VASSSVTYMY) of SEQ IDNO: 28, wherein one, two or three of these amino acid residues may besubstituted with a different amino acid; and/or a CDRL2 sequence thatcorresponds to amino acid residues 49 to 54 (THPLAS) of SEQ ID NO: 28,wherein one or two of these amino acid residues may be substituted witha different amino acid; and/or a CDRL3 sequence that corresponds toamino acid residues 87 to 95 (PHWNTNPPT) of SEQ ID NO: 28, wherein oneor two of these amino acid residues may be substituted with a differentamino acid.

The 2F5 antibody has a heavy chain as shown in SEQ ID NO: 31 and a lightchain as shown in SEQ ID NO: 32. An antibody of the invention maycomprise this variable heavy chain sequence and/or this variable lightchain sequence. The 2F5 antibody has the CDR sequences shown at aminoacids 31 to 35, 50 to 66 and 99 to 109 of SEQ ID NO: 31 and amino acids24 to 33, 49 to 55 and 88 to 96 of SEQ ID NO: 32. An antibody of theinvention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

An antibody according to the invention may comprise: a CDRH1 sequencethat corresponds to amino acid residues 31 to 35 (DYYMY) of SEQ ID NO:31, wherein one of these amino acid residues may be substituted by adifferent amino acid residue; and/or a CDRH2 sequence that correspondsto amino acids 50 to 66 (AISDDSTYTYYPDSVKG) of SEQ ID NO: 31, whereinone, two or three of these amino acids may be substituted by a differentamino acid residue; and/or a CDRH3 sequence that corresponds to aminoacid residues 99 to 109 (GGYGNLYAMDY) of SEQ ID NO: 31, wherein one, twoor three of these amino acid residues may be substituted by a differentamino acid.

An antibody according to the invention may comprise: a CDRL1 sequencethat corresponds to amino acid residues 24 to 35 (TASSSVSSSYLH) of SEQID NO: 32, wherein one, two or three of these amino acid residues may besubstituted with a different amino acid; and/or a CDRL2 sequence thatcorresponds to amino acid residues 51-57 (STSNLAS) of SEQ ID NO: 32,wherein one or two of these amino acid residues may be substituted witha different amino acid; and/or a CDRL3 sequence that corresponds toamino acid residues 90-98 (HQYHRSPFT) of SEQ ID NO: 32, wherein one ortwo of these amino acid residues may be substituted with a differentamino acid.

The 2F7 antibody has a heavy chain as shown in SEQ ID NO: 35 and a lightchain as shown in SEQ ID NO: 36. An antibody of the invention maycomprise this variable heavy chain sequence and/or this variable lightchain sequence. The 2F5 antibody has the CDR sequences shown at aminoacids 31 to 35, 50 to 66 and 99 to 109 of SEQ ID NO: 35 and amino acids24 to 34, 50 to 56 and 89 to 96 of SEQ ID NO: 36. An antibody of theinvention may comprise 1, 2, 3, 4, 5 or all 6 of these CDR sequences.

An antibody according to the invention may comprise: a CDRH1 sequencethat corresponds to amino acid residues 31 to 35 (NYVMH) of SEQ ID NO:35, wherein one of these amino acid residues may be substituted by adifferent amino acid residue; and/or a CDRH2 sequence that correspondsto amino acids 50 to 66 (WINPFNDGTNYNENFKN) of SEQ ID NO: 35, whereinone, two or three of these amino acids may be substituted by a differentamino acid residue; and/or a CDRH3 sequence that corresponds to aminoacid residues 99 to 109 (SGFITTLIEDY) of SEQ ID NO: 35, wherein one, twoor three of these amino acid residues may be substituted by a differentamino acid.

An antibody according to the invention may comprise: a CDRL1 sequencethat corresponds to amino acid residues 24 to 34 (KASESVGSFVS) of SEQ IDNO: 36, wherein one, two or three of these amino acid residues may besubstituted with a different amino acid; and/or a CDRL2 sequence thatcorresponds to amino acid residues 50 to 56 (GASNRYT) of SEQ ID NO: 36,wherein one or two of these amino acid residues may be substituted witha different amino acid; and/or a CDRL3 sequence that corresponds toamino acid residues 89 to 96 (GQYYTHPT) of SEQ ID NO: 36, wherein one ortwo of these amino acid residues may be substituted with a differentamino acid.

The term “antigen” (Ag) refers to the molecular entity used to immunisean immunocompetent vertebrate to produce the antibody (Ab) thatrecognizes the Ag. Herein, Ag is termed more broadly and is generallyintended to include target molecules that are specifically recognized bythe Ab, thus including fragments or mimics of the molecule used in theimmunization process, or other process, e.g. phage display, used forgenerating the Ab.

The term “epitope”, as used herein, is defined in the context of amolecular interaction between an “antigen binding polypeptide”, such asan antibody (Ab), and its corresponding antigen (Ag). Generally,“epitope” refers to the area or region on an Ag to which an Abspecifically binds, i.e. the area or region in physical contact with theAb. Physical contact may be defined using various criteria (e.g., adistance cut-off of 2-6 Å, such as 3 Å, such as 4 Å, such as 5 Å; orsolvent accessibility) for atoms in the Ab and Ag molecules. A proteinepitope may comprise amino acid residues in the Ag that are directlyinvolved in binding to a Ab (also called the immunodominant component ofthe epitope) and other amino acid residues, which are not directlyinvolved in binding, such as amino acid residues of the Ag which areeffectively blocked by the Ab, i.e. amino acid residues within the“solvent-excluded surface” and/or the “footprint” of the Ab.

The term epitope herein comprises both types of binding region in anyparticular region of PGLYRP1 that specifically binds to a PGLYRP1antibody. PGLYRP1 may comprise a number of different epitopes, which mayinclude, without limitation, conformational epitopes which consist ofone or more non-contiguous amino acids located near each other in themature PGLYRP1 conformation and post-translational epitopes whichconsist, either in whole or part, of molecular structures covalentlyattached to PGLYRP1, such as carbohydrate groups. PGLYRP1 may alsocomprise linear epitopes.

The epitope for a given antibody (Ab)/antigen (Ag) pair can be describedand characterized at different levels of detail using a variety ofexperimental and computational epitope mapping methods. The experimentalmethods include mutagenesis, X-ray crystallography, Nuclear MagneticResonance (NMR) spectroscopy, Hydrogen deuterium eXchange MassSpectrometry (HX-MS) and various competition binding methods; methodsthat are known in the art. As each method relies on a unique principle,the description of an epitope is intimately linked to the method bywhich it has been determined. Thus, depending on the epitope mappingmethod employed, the epitope for a given Ab/Ag pair may be describeddifferently.

At its most detailed level, the epitope for the interaction between theAg and the Ab can be described by the spatial coordinates defining theatomic contacts present in the Ag-Ab interaction, as well as informationabout their relative contributions to the binding thermodynamics. At aless detailed level, the epitope can be characterized by the spatialcoordinates defining the atomic contacts between the Ag and Ab. At aneven less detailed level the epitope can be characterized by the aminoacid residues that it comprises as defined by a specific criteria suchas the distance between or solvent accessibility of atoms in the Ab:Agcomplex. At a further less detailed level the epitope can becharacterized through function, e.g. by competition binding with otherAbs. The epitope can also be defined more generically as comprisingamino acid residues for which substitution by another amino acid willalter the characteristics of the interaction between the Ab and Ag.

In the context of an X-ray derived crystal structure defined by spatialcoordinates of a complex between an Ab, e.g. a Fab fragment, and its Ag,the term epitope is herein, unless otherwise specified or contradictedby context, specifically defined as PGLYRP1 residues characterized byhaving a heavy atom (i.e. a non-hydrogen atom) within a distance of,eg., 2-6 Å, such as 3 Å, such as 4 Å, such as 5 Å from a heavy atom inthe Ab.

From the fact that descriptions and definitions of epitopes, dependenton the epitope mapping method used, are obtained at different levels ofdetail, it follows that comparison of epitopes for different Abs on thesame Ag can similarly be conducted at different levels of detail.

Epitopes described at the amino acid level, e.g. determined from anX-ray structure, are said to be identical if they contain the same setof amino acid residues, Epitopes are said to overlap if at least oneamino acid is shared by the epitopes. Epitopes are said to be separate(unique) if no amino acid residue are shared by the epitopes.

The definition of the term “paratope” is derived from the abovedefinition of “epitope” by reversing the perspective. Thus, the term“paratope” refers to the area or region on the Ab to which an Agspecifically binds, i.e. with which it makes physical contact to the Ag.

In the context of an X-ray derived crystal structure, defined by spatialcoordinates of a complex between an Ab, such as a Fab fragment, and itsAg, the term paratope is herein, unless otherwise specified orcontradicted by context, specifically defined as Ag residuescharacterized by having a heavy atom (i.e. a non-hydrogen atom) within adistance of 4 Å from a heavy atom in PGLYRP1.

The epitope and paratope for a given antibody (Ab)/antigen (Ag) pair maybe identified by routine methods. For example, the general location ofan epitope may be determined by assessing the ability of an antibody tobind to different fragments or variant PGLYRP1 polypeptides. Thespecific amino acids within PGLYRP1 that make contact with an antibody(epitope) and the specific amino acids in an antibody that make contactwith PGLYRP1 (paratope) may also be determined using routine methods.For example, the antibody and target molecule may be combined and theAb:Ag complex may be crystallised. The crystal structure of the complexmay be determined and used to identify specific sites of interactionbetween the antibody and its target.

Antibodies that bind to the same antigen can be characterised withrespect to their ability to bind to their common antigen simultaneouslyand may be subjected to “competition binding”/“binning”. In the presentcontext, the term “binning” refers to a method of grouping antibodiesthat bind to the same antigen. “Binning” of antibodies may be based oncompetition binding of two antibodies to their common antigen in assaysbased on standard techniques such as surface plasmon resonance (SPR),ELISA or flow cytometry.

An antibody's “bin” is defined using a reference antibody. If a secondantibody is unable to bind to an antigen at the same time as thereference antibody, the second antibody is said to belong to the same“bin” as the reference antibody. In this case, the reference and thesecond antibody competitively bind the same part of an antigen and arecoined “competing antibodies”. If a second antibody is capable ofbinding to an antigen at the same time as the reference antibody, thesecond antibody is said to belong to a separate “bin”. In this case, thereference and the second antibody do not competitively bind the samepart of an antigen and are coined “non-competing antibodies”.

Antibody “binning” does not provide direct information about theepitope. Competing antibodies, i.e. antibodies belonging to the same“bin” may have identical epitopes, overlapping epitopes or even separateepitopes. The latter is the case if the reference antibody bound to itsepitope on the antigen takes up the space required for the secondantibody to contact its epitiope on the antigen (“steric hindrance”).Non-competing antibodies generally have separate epitopes.

An antibody according to the current invention may be capable ofcompeting with 1F10 for binding to PGLYRP1. An antibody according to thecurrent invention may be capable of competing with 1F36/mAb 0182 forbinding to PGLYRP1. An antibody according to the current invention maybe capable of competing with 1F95 for binding to PGLYRP1. An antibodyaccording to the current invention may be capable of competing with1F105/mAb 0184 for binding to PGLYRP1. An antibody according to thecurrent invention may be capable of competing with 2F5 for binding toPGLYRP1. An antibody according to the current invention may be capableof competing with 2F7 for binding to PGLYRP1. Hence, an antibodyaccording to the current invention may belong to the same bin as any oneor more of these antibodies.

The term “binding affinity” herein refers to a measurement of thestrength of a non-covalent interaction between two molecules, e.g. anantibody, or fragment thereof, and an antigen. The term “bindingaffinity” is used to describe monovalent interactions (intrinsicactivity).

Binding affinity between two molecules, e.g. an antibody, or fragmentthereof, and an antigen, through a monovalent interaction may bequantified by determining the equilibrium dissociation constant (K_(D)).In turn, K_(D) can be determined by measurement of the kinetics ofcomplex formation and dissociation, e.g. by the SPR method. The rateconstants corresponding to the association and the dissociation of amonovalent complex are referred to as the association rate constantk_(a) (or k_(on)) and dissociation rate constant k_(d) (or k_(off)),respectively. K_(D) is related to k_(a), and k_(d) through the equationK_(D)=k_(d)/k_(a).

Following the above definition, binding affinities associated withdifferent molecular interactions, such as comparison of the bindingaffinity of different antibodies for a given antigen, may be compared bycomparison of the K_(D) values for the individual antibody/antigencomplexes.

A PGLYRP1 antibody of the invention may have a K_(D) for its target(PGLYRP1) of 1×10⁻⁶M or less, 1×10⁻⁷ M or less, 1×10⁻⁸M or less, or1×10⁻⁹M or less, or 1×10⁻¹⁰M or less, 1×10⁻¹¹M or less, 1×10⁻¹²M or lessor 1×10⁻¹³M or less.

An antibody according to the current invention may be able to competewith another molecule, such as a naturally occurring ligand or receptoror another antibody, for binding to PGLYRP1. Therefore, an antibodyaccording to the current invention may be able to bind PGLYRP1 with agreater affinity that that of another molecule also capable of bindingPGLYRP1. The ability of an antibody to compete with a naturalligand/receptor for binding to an antigen may be assessed by determiningand comparing the K_(D) value for the interactions of interest, such asa specific interaction between an antibody and an antigen, with that ofthe K_(D) value of an interaction not of interest.

The term “binding specificity” herein refers to the interaction of amolecule such as an antibody, or fragment thereof, with a singleexclusive antigen, or with a limited number of highly homologousantigens (or epitopes). Antibodies that are capable of specificallybinding to PGLYRP1 are not capable of binding dissimilar molecules.Antibodies according to the invention may not be able to bind PGLYRPfamily members such as PGLYRP2, PGLYRP3 and PGLYRP4. Antibodiesaccording to the invention may not be able to bind human PGLYRP familymembers such as human PGLYRP2, human PGLYRP3 and human PGLYRP4.

The specificity of an interaction and the value of an equilibriumbinding constant can be determined directly by well-known methods.Standard assays to evaluate the ability of ligands (such as antibodies)to bind their targets are known in the art and include, for example,ELISAs, Western blots, RIAs, and flow cytometry analysis. The bindingkinetics and binding affinity of the antibody also can be assessed bystandard assays known in the art, such as SPR.

A competitive binding assay can be conducted in which the binding of theantibody to the target is compared to the binding of the target byanother ligand of that target, such as another antibody.

In another aspect, the present invention provides compositions andformulations comprising molecules of the invention, such as the PGLYRP1antibodies, polynucleotides, vectors and cells described herein. Forexample, the invention provides a pharmaceutical composition thatcomprises one or more PGLYRP1 antibodies of the invention, formulatedtogether with a pharmaceutically acceptable carrier.

Accordingly, one object of the invention is to provide a pharmaceuticalformulation comprising such a PGLYRP1 antibody which is present in aconcentration from 0.25 mg/ml to 250 mg/ml, and wherein said formulationhas a pH from 2.0 to 10.0. The formulation may further comprise one ormore of a buffer system, a preservative, a tonicity agent, a chelatingagent, a stabiliser, or a surfactant, as well as various combinationsthereof. The use of preservatives, isotonic agents, chelating agents,stabilisers and surfactants in pharmaceutical compositions is well-knownto the skilled person. Reference may be made to Remington: The Scienceand Practice of Pharmacy, 19^(th) edition, 1995.

In one embodiment, the pharmaceutical formulation is an aqueousformulation. Such a formulation is typically a solution or a suspension,but may also include colloids, dispersions, emulsions, and multi-phasematerials. The term “aqueous formulation” is defined as a formulationcomprising at least 50% w/w water. Likewise, the term “aqueous solution”is defined as a solution comprising at least 50% w/w water, and the term“aqueous suspension” is defined as a suspension comprising at least 50%w/w water.

In another embodiment, the pharmaceutical formulation is a freeze-driedformulation, to which the physician or the patient adds solvents and/ordiluents prior to use.

In a further aspect, the pharmaceutical formulation comprises an aqueoussolution of such an antibody, and a buffer, wherein the antibody ispresent in a concentration from 1 mg/ml or above, and wherein saidformulation has a pH from about 2.0 to about 10.0.

The PGLYRP1 antibodies of the present invention and pharmaceuticalcompositions comprising such antibodies may be used for the treatment ofinflammatory diseases such as the following: inflammatory bowel disease(IBD), Crohns disease (CD), ulcerative colitis (UC), irritable bowelsyndrome, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis,systemic lupus erythematosus (SLE), lupus nephritis, type I diabetes,Grave's disease, multiple sclerosis (MS), autoimmune myocarditis,Kawasaki disease, coronary artery disease, chronic obstructive pulmonarydisease, interstitial lung disease, autoimmune thyroiditis, scleroderma,systemic sclerosis, osteoarthritis, atoptic dermatitis, vitiligo, graftversus host disease, Sjogrens's syndrome, autoimmune nephritis,Goodpasture's syndrome, chronic inflammatory demyelinatingpolyneuropathy, allergy, asthma and other autoimmune diseases that are aresult of either acute or chronic inflammation. The PGLYRP1 antibodiesof the present invention and pharmaceutical compositions comprising suchantibodies may be used for the treatment of cardiovascular disease,stroke, ischemic reperfusion injury, pneumonia, sepsis and cancer.

PGLYRP1 antibodies of the invention are suitable for use in thetreatment of individuals with inflammatory bowel disease. InflammatoryBowel Disease (IBD) is a disease that may affect any part of thegastrointestinal tract from mouth to anus, causing a wide variety ofsymptoms. IBD primarily causes abdominal pain, diarrhea (which may bebloody), vomiting, or weight loss, but may also cause complicationsoutside of the gastrointestinal tract such as skin rashes, arthritis,inflammation of the eye, fatigue, and lack of concentration. Patientswith IBD can be divided into two major classes, those with ulcerativecolitis (UC) and those with Crohn's disease (CD). While CD generallyinvolves the ileum and colon, it can affect any region of the intestinebut is often discontinuous (focused areas of disease spread throughoutthe intestine), UC always involves the rectum (colonic) and is morecontinuous. In CD, the inflammation is transmural, resulting inabscesses, fistulas and strictures, whereas in UC, the inflammation istypically confined to the mucosa. There is no known pharmaceutical orsurgical cure for Crohn's disease, whereas some patients with UC can becured by surgical removal of the colon. Treatment options are restrictedto controlling symptoms, maintaining remission and preventing relapse.Efficacy in inflammatory bowel disease in the clinic may be measured asa reduction in the Crohn's Disease Activity Index (CDAI) score for CDwhich is scoring scale based on laboratory tests and a quality of lifequestionnaire. In animal models, efficacy is mostly measured by increasein weight and also a disease activity index (DAI), which is acombination of stool consistency, weight and blood in stool.

PGLYRP1 antibodies of the invention are suitable for use in thetreatment of individuals with rheumatoid arthritis. Rheumatoid arthritis(RA) is a systemic disease that affects nearly if not all of the bodyand is one of the most common forms of arthritis. It is characterized byinflammation of the joint, which causes pain, stiffness, warmth, rednessand swelling. This inflammation is a consequence of inflammatory cellsinvading the joints, and these inflammatory cells release enzymes thatmay digest bone and cartilage. As a result, this inflammation can leadto severe bone and cartilage damage and to joint deterioration andsevere pain amongst other physiologic effects. The involved joint canlose its shape and alignment, resulting in pain and loss of movement.

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop an inflammatory arthritis that resembles human rheumatoidarthritis. Since CIA shares similar immunological and pathologicalfeatures with RA, this makes it a suitable model for screening potentialhuman anti-inflammatory compounds. Efficacy in this model is measured bydecrease in joint swelling. Efficacy in RA in the clinic is measured bythe ability to reduce symptoms in patients which is measured as acombination of joint swelling, erythrocyte sedimentation rate,C-reactive protein levels and levels of serum factors, such asanti-citrullinated protein antibodies.

PGLYRP1 antibodies of the invention are suitable for use in thetreatment of individuals with psoriasis. Psoriasis is a T-cell mediatedinflammatory disorder of the skin that can cause considerablediscomfort. It is a disease for which there is currently no cure andaffects people of all ages. Although individuals with mild psoriasis canoften control their disease with topical agents, more than one millionpatients worldwide require ultraviolet light treatments or systemicimmunosuppressive therapy. Unfortunately, the inconvenience and risks ofultraviolet radiation and the toxicities of many therapies limit theirlong-term use. Moreover, patients usually have recurrence of psoriasis,and in some cases rebound shortly after stopping immunosuppressivetherapy. A recently developed model of psoriasis based on the transferof CD4+ T cells mimics many aspects of human psoriasis and therefore canbe used to identify compounds suitable for use in treatment of psoriasis(Davenport et al., Internat. Immunopharmacol 2: 653-672, 2002), Efficacyin this model is a measured by reduction in skin pathology using ascoring system. Similarly, efficacy in patients is measured by adecrease in skin pathology.

PGLYRP1 antibodies of the invention are suitable for use in thetreatment of individuals with psoriatic arthritis. Psoriatic arthritis(PA) is a type of inflammatory arthritis that occurs in a subset ofpatients with psoriasis. In these patients, the skin pathology/symptomsare accompanied by joint swelling, similar to that seen in rheumatoidarthritis. It features patchy, raised, red areas of skin inflammationwith scaling. Psoriasis often affects the tips of the elbows and knees,the scalp, the navel and around the genital areas or anus. Approximately10% of patients who have psoriasis also develop an associatedinflammation of their joints.

The term “treatment”, as used herein, refers to the medical therapy ofany human or other animal subject in need thereof. Said subject isexpected to have undergone physical examination by a medical orveterinary medical practitioner, who has given a tentative or definitivediagnosis which would indicate that the use of said treatment isbeneficial to the health of said human or other animal subject. Thetiming and purpose of said treatment may vary from one individual toanother, according to many factors, such as the status quo of thesubject's health. Thus, said treatment may be prophylactic, palliative,symptomatic and/or curative.

In terms of the present invention, prophylactic, palliative, symptomaticand/or curative treatments may represent separate aspects of theinvention.

An antibody of the invention may be administered parenterally, such asintravenously, such as intramuscularly, such as subcutaneously.Alternatively, an antibody of the invention may be administered via anon-parenteral route, such as perorally or topically. An antibody of theinvention may be administered prophylactically. An antibody of theinvention may be administered therapeutically (on demand).

Exemplary Embodiments

-   1. A cell expressing TREM-1, a signalling protein for TREM-1 and a    reporter construct that is activated by said signalling protein.-   2. The cell according to embodiment 1, wherein the cell is of    haematopoetic origin.-   3. The cell according to embodiment 2, wherein the cell is a myeloid    cell.-   4. The cell according to embodiment 2, wherein the cell is a T-cell.-   5. The cell according to any one of embodiments 1-4, wherein the    signalling protein is DAP10.-   6. The cell according to any one of embodiments 1-4, wherein the    signalling protein is DAP12.-   7. The cell according to any one of embodiments 1-4, wherein the    signalling protein is TCR zeta.-   8. The cell according to any one of embodiments 1-4, wherein the    signalling protein is Fc gamma RIII.-   9. The cell according to any one of embodiments 1-4, wherein the    signalling protein is a Fc receptor.-   10. The cell according to any one of embodiments 1-9, wherein the    reporter construct comprises a transcription factor and a reporter    gene.-   11. The cell according to embodiment 10, wherein said transcription    factor is NEAT.-   12. The cell according to embodiment 11, wherein said transcription    factor is NFkB.-   13. The cell according to any one of embodiments 10-12, wherein said    reporter gene encodes 3-galactosidase.-   14. The cell according to any one of embodiments 10-12, wherein said    reporter gene encodes luciferase.-   15. The cell according to any one of embodiments 10-12, wherein said    reporter gene encodes green fluorescent protein (GFP).-   16. The cell according to any one of embodiments 10-12, wherein said    reporter gene is a gene that encodes chloramphenicol transferase.-   17. The cell according to any one of embodiments 1-4, 6, 10-11 and    13, which is a BWZ.36/hTREM-1:DAP12:NFAT-LacZ T-cell.-   18. A method of stimulating the cell according to any one of    embodiments 1-17, comprising contacting said cell with PGLYRP1.-   19. A method of stimulating the cell according to any one of    embodiments 1-17, comprising contacting said cell with PGLYRP1 and    PGN.-   20. The method according to any one of embodiments 18-19, wherein    said PGLYRP1 is expressed by a cell.-   21. The method according to embodiment 20, wherein the cell    expressing PGLYRP1 is a prokaryotic cell.-   22. The method according to embodiment 20, wherein the cell    expressing PGLYRP1 is a eukaryotic cell.-   23. The method according to embodiment 22, wherein the cell    expressing PGLYRP1 is a mammalian cell.-   24. The method according to embodiment 23, wherein the cell    expressing PGLYRP1 is an activated neutrophil.-   25. The method according to embodiment 23, wherein the cell    expressing PGLYRP1 is a HEK cell.-   26. A method of identifying a TREM-1 ligand, comprising; (a)    culturing the cell according to any one of embodiments 1-18; (b)    detecting, preferably quantifying, the activity of said cell    expressing TREM-1 when it is contacted with a cell, a fluid such as    a biological fluid or a tissue that triggers TREM-1 activation; (c)    contacting the culture of (b) with a TREM-1 protein; (d) isolating    the component that binds TREM-1 and (e) characterising the isolated    component.-   27. A method of identifying a molecule that specifically binds    PGLYRP1 and that modifies TREM-1 mediated cellular activity,    comprising: (a) culturing the cell according to any one of    embodiments 1-18; (b) detecting, preferably quantifying, the    activity of said cell expressing TREM-1 when it is contacted with    PGLYRP1 and, optionally, a multimerisation agent such as PGN; (c)    contacting the culture of (b) with a molecule that specifically    binds PGLYRP1; and (d) detecting, preferably quantifying, that the    activity of said cell expressing TREM-1 is less than or more than    its activity as measured in (b).-   28. A method of identifying a PGLYRP1 antibody, or fragment thereof,    that modifies TREM-1 mediated cellular activity, comprising: (a)    culturing the cell according to any one of embodiments 1-18; (b)    detecting, preferably quantifying, the activity of said cell    expressing TREM-1 when it is contacted with PGLYRP1 and, optionally,    a multimerisation agent such as PGN; (c) contacting the culture    of (b) with an antibody that binds PGLYRP1; and (d) detecting,    preferably quantifying, that the activity of said cell expressing    TREM-1 is less than or more than its activity as measured in (b).-   29. The method according to embodiment 27, wherein said PGLYRP1    antibody, or fragment thereof, decreases TREM-1 mediated cellular    activity and wherein the activity of the first cell when measured    in (d) is less than its activity when measured in (b).-   30. The method according to embodiment 27, wherein said PGLYRP1    antibody, or fragment thereof, increases TREM-1 mediated cellular    activity and wherein the activity of the first cell when measured    in (d) is more than its activity when measured in (b).-   31. The PGLYRP1 antibody or fragment thereof that is identified by    means of the method according to any one of embodiments 28-30,-   32. A PGLYRP1 antibody or fragment thereof which is capable of    specifically binding PGLYRP1 and reducing PGLYRP1-mediated TREM-1    activity.-   33. The antibody, or fragment thereof, according to any one of    embodiments 31-32, which is capable of reducing the release of one    or more cytokines from cells that express TREM-1.-   34. The antibody or fragment thereof according to any one of    embodiments 31-33, which is a monoclonal antibody.-   35. The antibody or fragment thereof according to any one of    embodiments 31-34, which is a humanised antibody.-   36. The antibody or fragment thereof according to any one of    embodiments 31-34, which is a human antibody.-   37. The antibody or fragment thereof according to any one of    embodiments 31-34, which is a chimeric antibody.-   38. The antibody according to any one of embodiments 31-37, wherein    the isotype of said antibody is IgG.-   39. The antibody according to embodiment 38, wherein said isotype is    IgG1, IgG2 or IgG4.-   40. The antibody according to embodiment 37, wherein the isotype of    said antibody is IgG1.-   41. The antibody according to embodiment 37, wherein the isotype of    said antibody is IgG4.-   42. The antibody or fragment thereof according to any one of    embodiments 31-41, which is capable of competing with antibody 1F10    for binding to PGLYRP1.-   43. The antibody or fragment thereof according to any one of    embodiments 31-41, which is capable of competing with antibody    1F36/mAb 0182 for binding to PGLYRP1.-   44. The antibody or fragment thereof according to any one of    embodiments 31-41, which is capable of competing with antibody 1F95    for binding to PGLYRP1.-   45. The antibody according to any one of embodiments 31-41, which is    capable of competing with antibody 1F105/mAb 0184 for binding to    PGLYRP1.-   46. The antibody according to any one of embodiments 31-41, which is    capable of competing with antibody 2F5 for binding to PGLYRP1.-   47. The antibody according to any one of embodiments 31-41, which is    capable of competing with antibody 2F7 for binding to PGLYRP1.-   48. The antibody or fragment thereof according to any one of    embodiments 31-47, which is capable of specifically binding SEQ ID    NO: 37 (Type II 1.0 PGLYRP1) and/or SEQ ID NO: 38 (Type II 2.0    PGLYRP1).-   49. The antibody or fragment thereof according to any one of    embodiments 31-48, which has a K_(D) for its target that is 1×10⁻⁶M    or less, 1×10⁻⁷M or less, 1×10⁻⁸M or less, or 1×10⁻⁹M or less, or    1×10⁻¹⁰M or less, 1×10⁻¹¹M or less, 1×10⁻¹²M or less or 1×10⁻¹³M or    less, when determined using surface plasmon resonance.-   50. The antibody according to any one of embodiments 31-49, the    heavy chain of which comprises: a CDRH1 sequence corresponding to    amino acid residues 31 to 35 (SYWMN) of SEQ ID NO: 15, wherein one    of said amino acid residues may be substituted by a different amino    acid residue; and/or a CDRH2 sequence corresponding to amino acids    50 to 66 (MIHPSDSETRLNQKFKD) of SEQ ID NO: 15, wherein one, two or    three of said amino acids may be substituted by a different amino    acid residue; and/or a CDRH3 sequence corresponding to amino acid    residues 98 to 108 (DYSDYDGFAY]) of SEQ ID NO: 15, wherein one, two    or three of said amino acid residues may be substituted by a    different amino acid.-   51. The antibody according to any one of embodiments 31-49, the    heavy chain of which comprises: a CDRH1 sequence corresponding to    amino acid residues 31 to 35 (DYNMY) of SEQ ID NO: 19, wherein one    of said amino acid residues may be substituted by a different amino    acid residue; and/or a CDRH2 sequence corresponding to amino acids    50 to 66 (YIDPYNGDTSYNQKFKG) of SEQ ID NO: 19, wherein one, two or    three of said amino acids may be substituted by a different amino    acid residue; and/or a CDRH3 sequence corresponding to amino acid    residues 99 to 109 (GDYGNPFYLDY) of SEQ ID NO: 19, wherein one, two    or three of said amino acid residues may be substituted by a    different amino acid.-   52. The antibody according to any one of embodiments 31-49, the    heavy chain of which comprises: a CDRH1 sequence corresponding to    amino acid residues 31 to 35 (DTYIH) of SEQ ID NO: 23, wherein one    of said amino acid residues may be substituted by a different amino    acid residue; and/or a CDRH2 sequence of amino acids 50 to 66    (RIDPANDDTKYDPNFQG) of SEQ ID NO: 23, wherein one, two or three of    said amino acids may be substituted by a different amino acid    residue; and/or a CDRH3 sequence of amino acid residues 99 to 108    (SDNSDSWFAY) of SEQ ID NO: 23, wherein one, two or three of said    amino acid residues may be substituted by a different amino acid.-   53. The antibody according to any one of embodiments 31-49, the    heavy chain of which comprises: a CDRH1 sequence corresponding to    amino acid residues 31 to 35 (DYNMH) of SEQ ID NO: 27, wherein one    of said amino acid residues may be substituted by a different amino    acid residue; and/or a CDRH2 sequence corresponding to amino acids    50 to 66 (YVDPYDGGTSSNQKFKG) of SEQ ID NO: 27, wherein one, two or    three of said amino acids may be substituted by a different amino    acid residue; and/or a CDRH3 sequence corresponding to amino acid    residues 99 to 106 (EVPYYFDY) of SEQ ID NO: 27, wherein one, two or    three of said amino acid residues may be substituted by a different    amino acid.-   54. The antibody according to any one of embodiments 31-49, the    heavy chain of which comprises: a CDRH1 sequence that corresponds to    amino acid residues 31 to 35 (DYYMY) of SEQ ID NO: 31, wherein one    of these amino acid residues may be substituted by a different amino    acid residue; and/or a CDRH2 sequence that corresponds to amino    acids 50 to 66 (AISDDSTYTYYPDSVKG) of SEQ ID NO: 31, wherein one,    two or three of these amino acids may be substituted by a different    amino acid residue; and/or a CDRH3 sequence that corresponds to    amino acid residues 99 to 109 (GGYGNLYAMDY) of SEQ ID NO: 31,    wherein one, two or three of these amino acid residues may be    substituted by a different amino acid.-   55. The antibody according to any one of embodiments 31-49, the    heavy chain of which comprises: a CDRH1 sequence that corresponds to    amino acid residues 31 to 35 (NYVMH) of SEQ ID NO: 35, wherein one    of these amino acid residues may be substituted by a different amino    acid residue; and/or a CDRH2 sequence that corresponds to amino    acids 50 to 66 (WINPFNDGTNYNENFKN) of SEQ ID NO: 35, wherein one,    two or three of these amino acids may be substituted by a different    amino acid residue; and/or a CDRH3 sequence that corresponds to    amino acid residues 99 to 109 (SGFITTLIEDY) of SEQ ID NO: 35,    wherein one, two or three of these amino acid residues may be    substituted by a different amino acid.-   56. The antibody according to any one of embodiments 50-55, the    light chain of which comprises: a CDRL1 sequence corresponding to    amino acid residues 24 to 34 (RASQSISDYLH) of SEQ ID NO: 16, wherein    one, two or three of said amino acid residues may be substituted    with a different amino acid; and/or a CDRL2 sequence corresponding    to amino acid residues 51 to 56 (ASQSIS) of SEQ ID NO: 16, wherein    one or two of said amino acid residues may be substituted with a    different amino acid; and/or a CDRL3 sequence corresponding to amino    acid residues 89 to 97 (QNGHSFPLT) of SEQ ID NO: 16, wherein one or    two of said amino acid residues may be substituted with a different    amino acid.-   57. The antibody according to any one of embodiments 50-55, the    light chain of which comprises: a CDRL1 sequence corresponding to    amino acid residues 24 to 33 (SVSSSVNYMY) of SEQ ID NO: 20, wherein    one, two or three of said amino acid residues may be substituted    with a different amino acid; and/or a CDRL2 sequence corresponding    to amino acid residues 49 to 55 (DTSKLPS) of SEQ ID NO: 20, wherein    one or two of said amino acid residues may be substituted with a    different amino acid; and/or a CDRL3 sequence corresponding to amino    acid residues 88 to 96 (QQWTSNPPT) of SEQ ID NO: 20, wherein one or    two of said amino acid residues may be substituted with a different    amino acid.-   58. The antibody according to any one of embodiments 50-55, the    light chain of which comprises: a CDRL1 sequence corresponding to    amino acid residues 24 to 33 (SVSSSVNFMN) of SEQ ID NO: 24, wherein    one, two or three of said amino acid residues may be substituted    with a different amino acid; and/or a CDRL2 sequence corresponding    to amino acid residues 49 to 55 (DTSKLAP) of SEQ ID NO: 24, wherein    one or two of said amino acid residues may be substituted with a    different amino acid; and/or a CDRL3 sequence corresponding to amino    acid residues 88 to 96 (HQWSSYSLT) of SEQ ID NO: 24, wherein one or    two of said amino acid residues may be substituted with a different    amino acid.-   59. The antibody according to any one of embodiments 50-55, the    light chain of which comprises: a CDRL1 sequence corresponding to    amino acid residues 24 to 33 (VASSSVTYMY) of SEQ ID NO: 28, wherein    one, two or three of said amino acid residues may be substituted    with a different amino acid; and/or a CDRL2 sequence corresponding    to amino acid residues 49 to 54 (THPLAS) of SEQ ID NO: 28, wherein    one or two of said amino acid residues may be substituted with a    different amino acid; and/or a CDRL3 sequence corresponding to amino    acid residues 87 to 95 (PHWNTNPPT) of SEQ ID NO: 28, wherein one or    two of said amino acid residues may be substituted with a different    amino acid.-   60. The antibody according to any one of embodiments 50-55, the    light chain of which comprises: a CDRL1 sequence that corresponds to    amino acid residues 24 to 35 (TASSSVSSSYLH) of SEQ ID NO: 32,    wherein one, two or three of these amino acid residues may be    substituted with a different amino acid; and/or a CDRL2 sequence    that corresponds to amino acid residues 51-57 (STSNLAS) of SEQ ID    NO: 32, wherein one or two of these amino acid residues may be    substituted with a different amino acid; and/or a CDRL3 sequence    that corresponds to amino acid residues 90-98 (HQYHRSPFT) of SEQ ID    NO: 32, wherein one or two of these amino acid residues may be    substituted with a different amino acid.-   61. The antibody according to any one of embodiments 50-55, the    light chain of which comprises: a CDRL1 sequence that corresponds to    amino acid residues 24 to 34 (KASESVGSFVS) of SEQ ID NO: 36, wherein    one, two or three of these amino acid residues may be substituted    with a different amino acid; and/or a CDRL2 sequence that    corresponds to amino acid residues 50 to 56(GASNRYT) of SEQ ID NO:    36, wherein one or two of these amino acid residues may be    substituted with a different amino acid; and/or a CDRL3 sequence    that corresponds to amino acid residues 89 to 96 (GQYYTHPT) of SEQ    ID NO: 36, wherein one or two of these amino acid residues may be    substituted with a different amino acid.-   62. The antibody according to any one of embodiments 31-61 for use    as a medicament.-   63. The antibody according to any one of embodiments 31-62 for use    in the treatment of an inflammatory disease,-   64. An antibody that is capable of specifically binding PGLYRP1 and    reducing PGLYRP1-mediated TREM-1 activity for use as a medicament,-   65. An antibody that is capable of specifically binding PGLYRP1 and    reducing PGLYRP1-mediated TREM-1 activity for use in the treatment    of an inflammatory disease.-   66. Use according to any one of embodiments 62-65, wherein the    inflammatory disease is an autoimmune disease.-   67. Use according to embodiment 66, wherein the autoimmune disease    is rheumatoid arthritis (RA).-   68. Use according to embodiment 66, wherein the autoimmune disease    is inflammatory bowel disease (IBD) and/or ulcerative colitis.-   69. Use according to embodiment 66, wherein the autoimmune disease    is psoriatic arthritis (PA).-   70. Use according to embodiment 62 or 64 in the treatment of    cardiovascular disease, stroke, ischemia reperfusion injury, sepsis    and/or cancer.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1: Generation of a BWZ.36 humanTREM-1:DAP12 Stable CellLine

The BWZ.36/hTREM-1:DAP12:NFAT-LacZ cell line (herein also referred to asthe “BWZ/hTREM-1 reporter cell”) was derived from BW5147 T cells (Musmusculus thymus lymphoma cell line, ATCC TIB-47, LGC Standards,Middelsex, UK) and contains a LacZ reporter construct regulated by fourcopies of the NFAT promoter element (see Karttunen, J. & Shastri, N.(1991) Proc. Natl. Acad. Sci. USA 88, 3972-3976 and Fiering, S.,Northrop, J. P., Nolan, G. P., Matilla, P., Crabtree, G. R. &Herzenberg, L. A. (1990) Genes Dev. 4, 1823-1834). TREM/DAP12/pMX-IRESvector (encoding 786 bp of TREM-1 from a Smal site to BamHI site usingTREM-1 cDNA (Gene Bank Ref. ID: NM_018643.2, Sino Biological Inc.,Beijing, China) as template and oligo 5′ TAGTAGGGATCCGCTGGTGCACAGGAAGG(SEQ ID NO: 51) and 5′ TAGTAGGCGGCCGCTTCGTGGGCCTAGGGTAC (SEQ ID NO: 52)as primers cloned into pIREShyg vector GenBank Accession #U89672 (Cat.no. 6061-1, Clontech Laboratories, Calif., USA) was transfected inPLAT-E packaging cell line (provided by W. Yokoyama, WashingtonUniversity; alternatively, Cat. no. RV-101, Cell Biolabs Inc,Bio-Mediator KY, Vantaa, Finland) using Superfect transfection reagent(Cat. no. 301305, Qiagen Nordic, Denmark), PLAT-E supernatantscontaining TREM/DAP12/pMX-IRES viral particles were used to infectBWZ.36 cells as follows: 2×10⁵ BWZ.36 cells were cultured in 6 wellplates and the medium was replaced with 1.5 ml of supernatant containingthe viral particles +8 mg/ml of polybrene. After 6-8 hours, 1.5 ml ofnormal medium was added to the plate and the cells were incubated for anadditional 24 hours. BWZ.36 cell lines stably expressing TREM-1 werestained with anti TREM-1 monoclonal antibody (clone 21C7; Bouchon et al,2000, J. Immunol vol. 164 page 4991-4995) and isolated by cell sorting.

Example 2: Creation of a Bioassay for the Identification of CellsExpressing a Ligand for TREM-1

A TREM-1 reporter cell line was generated by transfecting the NFAT-lacZbearing cell line BWZ.36 (Sanderson S, Int. Immun. 1994) with hTREM-1and DAP12 as described in Example 1. This BWZ.36/hTREM-1:DAP12:NFAT-LacZcell line (herein also referred to as a BWZ/hTREM-1 reporter cell) ishighly responsive to antibody-mediated cross linking of TREM-1, giving˜40-fold induction of the NFAT-driven LacZ production when stimulatedwith 1-10 μg/ml plate bound commercially available anti-TREM-1 antibody,as compared to the isotype control. NFAT-driven LacZ production in thereporter cells may be assayed using a luminescence based kit, Beta Glow™(Promega E4720, Madison, Wis.). Plates were coated with isotype controlor aTREM-1 MAB1278 (Conc 3 ug/ml in PBS, 100 ul/well) (R&D Systems,Minneapolis, USA) at 4 C for 16 hours or for 2 hr at 37° C., 5% CO2 andthe BWZ/hTREM-1 reporter cells were detached by adding 10 ml Versene(catalog number #15040, Gibco, Carlsbad Calif., USA), spun at 400 g for5 min and washed in PBS and media (RPMI-1640 w/o phenol red; cat. number11835, Gibco, Carlsbad Calif., USA) before adding to the coated plates(1×10⁶ cells/ml, 4×10⁴ cells/well) to total volume 100 ul and incubatedovernight (16-20 hours) at 37° C., 5% CO₂.

These TREM-1 responsive cells were used to identify cells expressingTREM-1 ligand. One such cell turned out to be neutrophils from the wholeblood. The neutrophils of healthy donors were purified by means ofFicoll and dextran sedimentation and stimulated with PGN (InVivogen,tlrl-pgnsa, San Diego, Calif., USA) overnight. Briefly, the BWZ/hTREM-1reporter cells were added to the activated neutrophil cultures in a 1:3ratio of reporter cell:neutrophils. The assay was run in Poly-D-Lysinecoated Black cell culture plates (#356640 from BD Biosciences, San Jose,Calif., USA). TREM-1 activation was read out after 24 hours of cultureusing the BetaGlo reagent (E4720 from Promega, Madison, Wis., USA) andluminescence measured using a TopCount Luminescence counter from PerkinElmer.

The vitro stimulated neutrophils possessed a ligand that was able toinduce TREM-1 signalling, and neutrophils from the whole blood ofhealthy donors were purified by Dextran sedimentation and stimulatedovernight with multiple reagents. The only reagent that was able tostimulate a TREM-1 responsive signal from neutrophils was PGN-SA(Invivogen, tlrl-pgnsa, San Diego, Calif., USA) which mimic bacterialactivation of the cells. These activated neutrophils were then used tostimulate the BWZ/hTREM-1 reporter cell line, by co-culturing the cells.Briefly, the BWZ/hTREM-1 reporter cells were added to the activatedneutrophil cultures in a 1:3 ratio of reporter cell:neutrophils. Theassay was run in Poly-D-Lysine coated black cell culture plates (cat.no. 356640 BD Biosciences, San Jose, Calif., USA), TREM-1 activation wasread out after 24 hours of culture using the BetaGlo reagent (cat. no.E4720, Promega, Madison, Wis., USA) and luminescence measured using aTopCount Luminescence counter from (Perkin Elmer, Waltham Mass., USA).As shown in FIG. 1, significant induction of reporter activity wasobserved in BWZ/hTREM-1 cells co-cultured with the PGN-stimulatedneutrophils. This induction was highly dependent on neutrophilactivation. No response was seen with resting neutrophils (bar 2neutrophils). Likewise, no response was seen when stimulatingBWZ/hTREM-1 reporter cells with PGN-SA in the TLRL cocktail in theabsence of neutrophils (bar 1-TLRL), demonstrating that the response isnot merely a direct effect of PGN on the BWZ/hTREM-1 cells. Activatingneutrophils with cytokine cocktails (bar 3+4 neutrophils+cytokines) didnot provide the correct TREM-1 activation signal either.

Example 3: Binding of Soluble TREM-1 to PGN-Activated Neutrophils

PGN-stimulated neutrophils were able to induce TREM-1 activation,indicating the presence of a TREM-1 stimulating factor in thePGN-stimulated neutrophil cultures. In order to confirm the presence ofa TREM-1-interacting protein on the neutrophils, PGN-stimulatedneutrophils were stained with a recombinant TREM-1-tetrameric proteinand analysed by flow cytometry. Briefly, granulocytes were isolated fromhuman whole blood obtained from Astarte Biologics (Redmond, Wash., USA)via a Ficoll-dextran sedimentation method. Blood was stratified onFicollPaque (17-0840-03, GE Healthcare, Piscataway, N.J., USA) gradientwith a ratio of 3 parts of Ficoll and 4 parts of blood in a 50m1 tube,then centrifuged at 400×g for 30 minutes at 22° C., without brake. Theintermediate PBMC band was gently removed by aspiration. Thegranulocytes stratified on the packed RBC were aspirated and transferredto a 50 ml polypropylene tube. The granulocytes and contaminating RBCswere diluted to 40 ml with 1x PBS and followed by addition of 10 ml 4%DEXTRAN 500 (Sigma, 31392, St Louis, Mo., USA) in PBS solution. Aftermixing by gentle inversion, the tubes were left at 22° C. for 20-30 min.A granulocyte rich supernatant was then transferred into a fresh tubeand centrifuged at 250×g, 5 min, 22° C.; the supernatant was aspiratedand discarded. Contaminating RBCs were removed with an osmotic lysis,briefly, the cell pellet was resuspended in 7.5 ml of 0.2% NaCl; gentlymixed for 55-60 seconds and 17.5 ml of a 1.2% NaCl. solution was added.The volume was then brought to 50 ml with PBS and spun at 250×g for 5min, the pellet was resuspended in 7.5 ml of 0.2% NaCl to repeat thelysis a second time. The final granulocyte pellet was resuspended inRPMI/10% FBS.

Isolated granulocytes were cultured at a density of 3.8 E6/ml inRPMI/10% FBS+10 μg/ml of PGN-SA (Invivogentlrl-pgnsa, San Diego, Calif.,USA) for 7 days. Cells were pelleted by centrifugation and resuspendedin PBS/2% FBS for staining. Resuspended granulocytes were then plated ina 96 well plate (round bottom) at a density of 100,000/well in thepresence or absence of 2 μg/ml of probe, with or without 100 μg/ml (50X)of a specific or irrelevant competitor protein. Cells were incubatedwith probe/competitor for 1 hour at 4 C in a volume of 50 μl/well. Atthe end of the incubation, 150 μl/well of PBS/2% FBS was added and thecells were pelleted. The pelleted cells were resuspended in 50 μl/wellof goat anti hFc F(ab′)2/PE conjugate (Jackson ImmunoResearch109-116-098, West Grove, Pa., USA) and incubated at 4° C. for 30minutes. 150 μl/well of PBS/2% FBS was added and the cells pelleted.Pelleted cells were further washed in 200 μl/well PBS/2% FBS andpelleted. Washed cells were then resuspended in 100 μl/well of fixative(1:1 PBS: Cytofix. 554655, BD Biosciences, San Jose, Calif., USA) andincubated 5 minutes at room temperature, 100 μl/well of PBS/2% FBS wasadded to fixed cells, then the cells were pelleted. The stained/fixedcells were then resuspended in 100 μl/well of PBS/2% FBS for flowcytometric analysis on a LSR II flow cytometer. (BD Biosciences, SanJose, Calif., USA).

Probe Set:

Fc mut 5.36 mg/ml SEQ ID NO: 3 hTREM-1 tet/Fc mut 1.07 mg/ml SEQ ID NO:2Competitors for hTREM-1 tet/Fc Mut:

50X DCIR COMP  0.3 mg/ml SEQ ID NO: 4 50X TREM COMP 1.14 mg/ml SEQ IDNO: 5

Histograms were created from the flow cytometric data. The backgroundfluorescence from the goat anti hFc/PE conjugate was shown in eachhistogram as the background and was designated “PE”. An identical markerwas drawn on each histogram to designate the percentage of cells thatbound to the secondary goat anti hFc/PE conjugated antibody. Thenegative control Fc mut protein showed 2% positive binding cells, whichwas identical to the background fluorescence seen with the goat antihFc/PE conjugate alone (FIG. 2A). When cells were stained with 2 μg/mlhTREM-1/tetramer they were 39% positive (FIG. 2B). Men the TREM-1tetramer binding was done in the context of 100 μg/ml DCIR COMP protein,the cells were 41% positive confirming that the DCIR COMP did notcompete for binding with TREM-1 tetramer (FIG. 2C). When the TREM-1tetramer binding was done in the presence of 100 μg/ml of TREM-1 COMP,the cells were 10% positive showing that the TREM COMP protein didcompete with TREM-1 tetramer for binding to the cells (FIG. 2D).

Example 4: Identification of PGLYRP1 as a Neutrophil-Expressed TREM-1Ligand

PGLYRP1 was identified as a TREM-1 ligand through the use ofimmunoprecipitation coupled with mass spectroscopy (IP-MS). SolubleTREM-1 tetramer was used as an affinity “bait” molecule to identify aligand. Briefly, TREM-1-tetramer-Fc (SEQ ID NO: 2) and separatelyCD83-Fc (SEQ ID NO: 5) were each incubated at final concentrations of100 μg/ml with 270 million human neutrophils, purified by dextransedimentation as described above, in 1 mL PBS at 4° C., 90 minutes withmild shaking. After pelleting, the cells were resuspended in 1 mL PBSbuffer with the inclusion of the crosslinker3,3′-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) (Thermo Scientific:21578, Rockford, Ill., USA), at a concentration of 2 mM and incubated 30minutes at room temperature. Cells were washed 3× with 1 mL PBS followedby lysis in 1 mL RIPA buffer (Thermo Scientific, 89901, Rockford, Ill.,USA). The lysate was centrifuged at 15,000×g for 10 minutes at 4° C. toremove insoluble materials. Neutrophil proteins cross-linked to Fccoupled probes were immunoprecipitated from the supernatant usingProtein A Mag Sepharose™ beads (GE Healthcare Life Sciences, 28-9670-56,Piscataway, N.J., USA). Briefly, 50 μL of beads were first washed with200 μL PBS, then resuspended in 1 mL of cell lysate, incubated 60minutes at 4° C., magnetically captured, and sequentially washed 2× with200 μl RIPA buffer then 3× with 200 μL PBS. Upon removing PBS from thefinal magnetic capture, proteins were eluted from the magnetic beadsusing 200 μL buffer containing 8 M Urea, 100 mM Tris (pH 8.0), and 15 mMTCEP (Thermo Scientific, 77720, Rockford, Ill., USA) and incubated atroom temperature for 30 minutes, beads were captured and supernatant wastransferred to a Microcon Ultracel YM-30 filter (Millipore, 42410,Billerica, Mass., USA). Samples were spun at 14,000×g, 20° C., 30-60minutes until no liquid remained on the top of the filter membrane. Theretained proteins were then alkylated with 100 μL 50 mM IAA(iodoacetamide) in 8 M Urea for 30 minutes in dark at room temperature.The filter was washed 2× with 100 μL 50 mM NH₄HCO₃ and then transferredto a new collection tube. 1 μg trypsin (cat. no. V5111, Promega, MadisonWis., USA) in 60 μL 50 mM NH₄HCO₃ was added followed by incubation at37° C. overnight. The tryptic digest was collected by centrifugation at14,000×g for 30 minutes followed by washing the filter with 50 μL 50 mMNH₄HCO₃. 10 μL of the digest was analyzed by LC/MS/MS using anLTQ-Orbitrap-XL mass spectrometer (Thermo Scientific, Waltham, Mass.,USA). The data was searched against IPI human database (v3.81) usingSEQUEST-Sorcerer engine (4.0.4 build) (SageN, Milpitas, Calif., USA) andthen post processed with Scaffold 3 (Proteome Software, Portland, Oreg.,USA) to filter protein IDs with a false discovery rate of 1%. Afternegative control subtraction, PGLYRP1 was found to be a high-confidenceprotein specifically associated with hTREM-1 tetramer. Theimmunoprecipitation in the neutrophils showed that out of the 148identified proteins, 72 proteins were immunoprecipitated by the controlconstruct (CD83) alone, 73 of the proteins were identical for TREM-1 andCD83, whereas only three were TREM-1 specific (FIG. 3A and FIG. 3B). Theexperiment was subsequently repeated using neutrophils from a differentdonor and PGLYRP1 was again identified as specifically interacting withhTREM-1.

Example 5: Purification of Human PGLYRP1 Expressed from HEK293 6E

A recombinant protein sequence was constructed by fusing the human CD33signal peptide sequence (SEQ ID NO: 53) with the human mature PGLYRP1coding sequence (SEQ ID NO: 1). The resulting open reading frame wascloned into pcDNA3.1/Zeo(+) vector (Life Technologies, Carlsbad Calif.,USA) after a CMV promoter. The pcDNA3.1-hPGLYRP1 construct was thentransfected into HEK293 6E cells with 293fectin™ (Life Technologies,Carlsbad Calif., USA) following the vendor's protocol. 5 days aftertransfection, the culture supernatant containing secreted human PGLYRP1was harvested by centrifugation (15,000 rpm×20 min, 4□C) and thencleared by the filtration with 0.22 μm cellulose nitrate membrane. Thecleared supernatant was first diluted 10 fold into 20 mM sodium citratepH 5.0 and then applied to a Hitrap SP HP 5 ml column (17-1151-01 GEHealthcare, Uppsala, Sweden), followed by a 5 column volume wash with 20mM sodium citrate pH5.0. The bound human PGLYRP1 was then eluted with a0˜100% linear gradient of 20 mM sodium citrate pH 5.0, 1M NaCl in 30column volumes. The fractions containing dimer and monomer forms ofhuman PGLYRP1 were pooled separately and concentrated to less than 4 mlby Amicon ultra 15 centrifugal units (UFC800324 3,000 kDa MWCO,Millipore, Hellerup, Denmark). Dimer and monomer pools were furtherpolished and buffer-exchanged to Phosphate Buffered Saline (PBS) by aHiload 26/60 Superdex 75 318 ml column (17-1070-01 GE Healthcare,Uppsala, Sweden). After concentrating, the final protein concentrationswere determined by measuring 280 nm absorbance with a NANODROP UVspectrometer. Protein purity was assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).

Example 6: Refolding and Purification of Human PGLYRP1 Expressed from E.coli

Human PGLYRP1 was expressed as inclusion bodies in Escherichia coli BL21(DE3) cells. Bacteria were harvested by centrifugation, resuspended in50 mM Tris-HCl pH8.0, 500 mM NaCl, 5 mM EDTA, 0.5% Triton X-100 anddisrupted by sonication. The insoluble pellet was washed three timeswith 50 mM Tris, pH 8.0, 1% TritonX-100, 2 M urea and once with 50 mMTris pH 8.0, then solubilized in 50 mM Tris-HCl. 6M guanidinehydrochloride, pH7.4, 1 mM DTT (final protein concentration 20 mg/ml).For in vitro folding, solubilized human PGLYRP1 inclusion bodies werediluted into 50 mM Tris, pH 8.0, 2 mM EDTA, 5 mM cysteamine, 0.5 mMcystamine, 0.4 M arginine (final protein concentration 1 mg/ml), Afterovernight at 4° C., the folding mixture was cleared bycentrifugation/filtration and then diluted 12 fold into 10 mM MES pH 3.5to lower the conductivity and pH (final pH ˜5.8, conductivity ˜6 mS/cm).The diluted folding mixture was then applied to a Hitrap SP HP 5 mlcolumn (17-1151-01 GE Healthcare, Uppsala, Sweden), followed by a 5column volume wash with 50 mM MES pH 5.8. The bound human PGLYRP1 wasthen eluted with a 0˜60% linear gradient of 50 mM MES pH 5.8, 1 M NaClin 20 column volume. The fractions containing refolded human PGLYRP1were pooled and concentrated to less than 4 ml by Amicon ultra 15centrifugal units (UFC800324 3,000 kDa MWCO, Millipore, Hellerup,Denmark). A Hiload 26/60 Superdex 75 318 ml column ((17-1070-01 GEHealthcare, Uppsala, Sweden)was then used to polish and buffer-exchangethe proteins to Phosphate Buffered Saline (PBS). Majority of refoldedhuman PGLYRP1 proteins was in monomer form. After concentrating, thefinal protein concentration was determined by measuring 280 nmabsorbance with a NANODROP UV spectrometer. Protein purity was assessedby sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Example 7: Immunisation of Mice and Identification of mAbs

Purified human PGLYRP1 was used to immunize mice in order to raiseantibodies. Briefly, mice were immunised 3 times with 20 μg recombinantPGLYRP1 per immunisation. The first immunisation was subcutaneous usingComplete Freunds Adjuvant (cat. no. 3018, Statens Serum Institut,Copenhagen, Denmark). The following two immunisations wereintraperitoneal using Incomplete Freunds Adjuvant (cat. no. 3016,Statens Serum Institut, Copenhagen, Denmark). Ten days after the lastimmunisation, cheek blood was drawn and the sera were tested againstPGLYRP1 in a direct ELISA.

Example 8: Binding of Soluble TREM-1 to PGLYRP1

One common method of validating a novel protein-protein interaction isthrough reconstituting the interaction using recombinant reagents. Tothis end recombinant human PGLYRP1 was expressed with a C-terminalepitope signalling the posttranslational addition of aglycosylphosphatidylinositol (GPI) structure. Proteins containingterminal GPI structures are targeted for display on the plasma membrane.This commonly applied technique allows otherwise soluble proteins to bedisplayed and tested for binding by flow cytometric techniques. In FIG.4A, HEK-2936E cells were transfected with pCDNA 3.1/zeo(+) hPGLYRP1-GPI(SEC ID NO: 7) 3 μg of DNA was diluted into 100 μl of Optimem (Cat. no.31985062, Life Technologies, Carlsbad, Calif., USA). 4 μl of 293fectin(Cat. no. 51-0031, Life Technologies, Carlsbad, Calif., USA) was addedto 100 μl of Optimem (Cat. no. 31985062, Life Technologies, Carlsbad,Calif., USA) and incubated at 22° C. for 5 minutes. The DNA/Optimem mixand the 293fectin/Optimem mix were combined and incubated for anadditional 20 minutes at room temperature. HEK-2936E cells were dilutedto 3 mls at 1e6/ml in Freestyle media (Cat. no. 12338, LifeTechnologies, Carlsbad, Calif., USA) and plated in a 6-well dish (Cat.no. 35-3046, Biosciences San Jose, Calif., USA) and then theDNA/293fectin mix was added dropwise to the cells. Cells were incubatedfor 48 hours at 37° C. with shaking. 1μg/ml of humanTREM-1-G4S×3-TREM-1/Fc6mut (SEQ ID NO: 2) was diluted in PBS/2% FBS and50 μl of probe was added to 80,000 transfected HEK293-6E cells in around bottom 96-well plate (Cat. no. 3799, Costar, Lowell, Mass., USA).Cells were incubated at 4° C. for 1 hr followed by 2× washes with 200 μlPBS/2% FBS. 50 .μl of 1 μg/ml PE goat anti-human Fc (Cat. no.109-116-098, Jackson lmmunoResearch, West Grove, Pa., USA) was added andincubated for an additional hour followed by 2× PBS/2% FBS washes. Cellswere fixed for 5 min in 1:1 PBS diluted BDCytofix (Cat. no. 554655, BDBiosciences, San Jose, Calif., USA) and incubated for 5 mins followed bya 200 μl PBS/2% FBS wash. Cells were resuspended in 100 μl PBS/2% FBSbefore being analyzed on a FACS LSRII (BD Biosciences, San Jose,Calif.). The results of this experiment are shown in FIG. 4A whereunstained cells are shown by solid black line (G03),TREM-1-G4S×3-TREM-1/Fc6mut staining of mock transfected cells is showndashed line, and shows no binding relative to unstained cells. Finally,cells transfected with human PGLYRP1-GPI robustly bindTREM-1-G4S×3-TREM-1/Fc6mut shown with the dotted line. Quantitation ofbinding is expressed as mean florescent intensity, MFI.

In addition to flow cytometry, protein-protein interactions are alsocommonly assessed by measuring surface plasmon resonance (SPR). ABiacoreT200 (GE Healthcare, Piscataway, N.J., USA) instrument was usedto analyze the interaction between human TREM-1 & human PGLYRP1 and alsobetween human PGLYRP1 &sonicated, soluble E. coli peptidoglycan (Cat.no, tlrl-ksspgn, Invivogen, San Diego, Calif., USA). All assays wereperformed at 25° C. at flow rates of 20-30 μL/minute in 1× HBS-P runningbuffer (Cat. no. BR-1006-71, GE Healthcare, Piscataway, N.J., USA).

In FIG. 4B, 4641.1 RU of human TREM-1 tetramer (SEQ ID NO: 2) was aminecoupled to a Biacore CM5 chip (Cat. no, BR-1005-30, BR-1000-50, GEHealthcare, Piscataway, N.J., USA) following the manufacturer'srecommended methods. PGLYRP1 (Cat. no. 2590-PG, R&D Systems,Minneapolis, USA) was diluted to 150 nM in 1× HBS-P running buffer (Cat.no. BR-1006-71, GE Healthcare, Piscataway, N.J., USA) in the presence orabsence of 10 μg/mL soluble E. coli peptidoglycan (Cat. no. tlrl-ksspgn,Invivogen, San Diego, Calif., USA) and injected over the TREM-1 surface.Data is reference subtracted vs, an activated and blocked (withethanolamine) reference surface. Though PGLYRP1 binds TREM-1 both inpresence and absence of PGN, there appears to be a significant avidityeffect when PGLYRP1 is crosslinked by PGN.

In FIG. 4C, 274.8 RU of PGLYRP1 dimer (SEQ ID NO: 1) were amine coupledto a Biacore CM5 chip (Cat. no. BR-1005-30, BR-1000-50, GE Healthcare,Piscataway, N.J.). Soluble E. coli peptidoglycan (Cat. no, tlrl-ksspgn,Invivogen, San Diego, Calif., USA) was diluted to 10 μg/mL in 1× HBS-Prunning buffer (Cat. no. BR-1006-71, GE Healthcare, Piscataway, N.J.,USA) and injected over the PGLYRP1 surface. Data is reference subtractedvs. an activated and blocked (with ethanolamine) reference surface.

In FIG. 4D, the same chip was used as in FIG. 4C, (274.8 RU of PGLYRP1dimer, (SEQ ID NO: 1). TREM-1 dimer (SEQ ID NO: 8) was diluted to 150 nMin 1× HBS-P running buffer (BR-1006-71, GE Healthcare, Piscataway, N.J.,USA) and injected both before and after repeated injections ofpeptidoglycan (depicted in FIG. B). Data is reference subtracted vs. anactivated and blocked (with ethanolamine) reference surface and thesignal of a buffer blank injection is also subtracted. TREM-1 showsclear binding to immobilized PGLYRP1. Soluble TREM-1 dimer bindssimilarly to immobilized PGLYRP1 alone or to PGLYRP1 that has beenloaded with PGN. Overall binding of surface immobilized TREM-1 receptorby soluble PGLYRP1 and PGN consists of a modest affinity for thePGLYRP1:TREM-1 complex enhanced by an avidity effect from the highlycrosslinked PGLYRP1.

Example 9: Activation of TREM-1-Response by Recombinant PGLYRP1

To test if recombinant PGLYRP1 can activate a TREM-1 response, theBWZ/hTREM-1 reporter cell line was seeded into black 96 well plates andstimulated with recombinant human PGLYRP1 (Cat, no. 2590-PG-050, R&DSystems: Minneapolis, Minn.) in the presence or absence of 10 μg/ml PGN.TREM-1 activation was read out after 24 hours of culture using theBetaGlo reagent (Cat. no. E4720, Promega Madison, Wis., USA) andluminescence measured using a TopCount Luminescence counter from PerkinElmer. As shown in FIG. 5A, stimulation of the TREM-1 reporter cell linewith PGLYRP1 induced a dose-dependent activation of TREM-1 in thepresence of PGN. Several different PGN were tested, including PGN-EC(from E. coli), PGN-SA (from S. aureus) and PGN-BS (from B. subtilis)(Invivogen, San Diego, Calif., USA), and were all able to fascilitatethe PGLYRP1-induced TREM-1 response.

The response induced by recombinant PGLYRP1 could be inhibited byaddition of a recombinant TREM-1-Fc-fusion protein (SEQ ID NO: 2) (FIG.5B) or by addition of polyclonal anti-PGLYRP1 antibody (Cat. no.AF-2590, R&D Systems: Minneapolis, Minn., USA) confirming that PGLYRP1is the TREM-1 ligand and that soluble TREM-1 and anti-PGLYRP1 arepotentially useful as TREM-1 antagonists.

Example 10: TNFalpha Release from M1 Macrophages Stimulated by PGLYRP1

Monocytes were differentiated into M1 macrophages and stimulated withPGLYRP1 complex resulting in TNFalpha release in two different donors.

Those skilled in the art will recognize the value of establishing afreezer bank collection of primary cells from multiple donors thusproviding for convenient replication of experiments. In vitro derivedmacrophages were produced from peripheral blood monocytes as follows.Negatively enriched monocytes were isolated from a peripheral blood“leukopak” obtained from Research Blood Components (Brighton, Mass.,USA) using a Rosette Sep kit (cat. no. Cat, no. 15068 Stem CellTechnologies, Vancouver BC, Canada) following the manufacturer'sinstructions. Isolated monocytes were suspended in 10% DMSO/FBS aliquotsof 50e6 cell/ml and gradually cooled to −80C. To produce macrophagecells, one or more frozen vials of monocytes were rapidly thawed in a 37C water bath, diluted to 10 ml with growth media [RPMI 1640 (Cat. no.72400-047, Life Technologies, Carlsbad Calif., USA)) with 10% FBS (Cat.no. 03-600-511, Themo Fisher, Waltham Mass., USA) and centrifuged 5minutes at 250g. Cells were suspended to 2e6 cells/ml in growth mediasupplemented with 50 ng/ml human MCSF (Cat. no. PHC9501, LifeTechnologies, Carlsbad Calif., USA), placed into tissue culture treated,petri style tissue culture plates and into a humidified incubatorprogrammed to maintain a “hypoxic” atmosphere of 5% CO2, 2% O2. On thethird day in culture, the cells were fed with the addition of an equalvolume of growth media supplemented with 50 ng/ml human MCSF. After 6days in culture the monocytes had differentiated into MO macrophages. M0cells were further differentiated by changing the media to growth mediasupplemented with 50 ng/ml human IFNg (Cat. no., PHC4031, LifeTechnologies, Carlsbad Calif., USA) for M1 macrophages or 40 ng/ml humanIL-4 (Cat. no. PHC0045, Life Technologies, Carlsbad Calif., USA) for M2macrophages and returning to the incubator for an additional 22 hours.On the seventh day, macrophages were suitably differentiated to be usedin a bioassay. Briefly, macrophages were recovered from the petri platesby washing with 1× PBS, followed by 5 mM EDTA in PBS. The plates werethen returned to 37 C for 30 minutes and cells were “power washed” offthe plate using a 10 ml syringe and 22 G needle. Cells were then dilutedinto growth media, centrifuged at 250 g for 5 minutes after which thecell pellet was suspended to a final concentration of 1e6/ml.

Macrophage cells prepared as above were used in bioassays wherecytokines such as TNF-alpha produced in response to stimulation of thecells with TREM-1 ligand were measured in the conditioned media byELISA. Such a bioassay was further utilized to measure blockade ofTREM-1 ligand stimulation by TREM-1 specific antibodies. TREM ligand ornegative controls were prepared at 4× concentrations in growth media and50 microliters/well were added to 96 well microtiter dishes. Finalconcentrations of TREM-1 ligand consisted of 7.5 ng/ml recombinant humanPGLYRP1 (generated as described in example 5) and 3 μg/ml PGN-BS (Cat.no. tlrl-pgnbs, Invivogen, San Diego Calif., USA). Cells were culturedunder humidified hypoxic conditions as described above for 22 hoursafter which conditioned media was collected and TNF-alpha levels weremeasured by ELISA, following manufacturer's instructions (Cat. no.DY210, R&D Systems, Minneapolis Minn., USA).

Donor 1 Donor 2 TNF-α, TNF-α, pg/ml pg/ml M1 macrophage with: Avg SD AvgSD No addition 0 0 22 1 0.4 μg/ml PGN-BS 357 153 764 139 2.0 μg/mlPGN-BS 3086 151 5792 226 5 μg/ml PGLYRP1 + 0.4 μg/ml 7502 384 7819 945PGN-BS 5 μg/ml PGLYRP1 + 2 μg/ml PGN-BS 31440 1030 40418 1633This example shows that the TREM-1 ligand PGLYRP1 is able to furtherincrease TNFa release from macrophages from two different donors.

Example 11: Cytokine Release from Synovial Tissue Cells from RA PatientsUpon Stimulation with PGLYRP1

Synovial tissue samples were obtained from RA patients during total kneereplacement. Single suspension of synovial tissue cells was isolated bya digestion via 4 mg/ml of collagenase (Cat, no. 11088793001, Roche,Mannheim, Germany) and 0.1 mg/ml of DNase (Cat. no. 11284932001, Roche,Mannheim, Germany) for 1 h at 37 degree.

Synovial tissue cells at 1×10̂5/well in culture medium RPMI (Cat, no,R0883, Sigma Aldrich, St Louis, Mo., USA)+10% FCS (Cat, no. S0115,BioChrom AG, Grand Island, N.Y. 14072, USA) were stimulated with 4 ug/mlof PGLYRP1 and 1 ug/ml of PGN-ECNDi (Cat. no. tlrl-kipgn, Invivogen, SanDiego, Calif. 92121, USA). After 24 h incubation, cell supernatants wereharvested, and cytokines were measured by either ELISA (TNFa (Cat, no,DY210, R&D Systems, Minneapolis, Minn. 55413 USA), IL-1b (Cat, no.88-7010-88, eBioscience, San Diego Calif. USA), GM-CSF (Cat. no.88-7339-88, eBioscience, San Diego Calif. USA) or Flowcytomix (TNFa,IL-1b, MIP-1b, MCP-1, IL-6, and IL-8 (Cat. no. BMS, eBioscience, SanDiego Calif. USA). The cytokines were secreted from the synovial tissuecells upon stimulation with the TREM-1 ligand.

Cytokine (pg/ml) PGN PGN + PGLYRP1 TNFalpha 623.69 1444.59 IL-1beta2419.42 3772.74 GM-CSF 181.91 615.91 MIP-1beta 2457.955 4394.725 MCP-1273.055 471.26 IL-6 2056.94 4189.355 IL-8 2574.56 5509.195This example shows that cells from synovial tissue from rheumatoidarthritis patients will respond to stimulation by the TREM-1 ligandPGLYRP1 by secreting numerous cytokines.

Example 12: Identification of Anti-PGLYRP1 mAbs that Inhibit TREM-1Activation

In order to identify monoclonal anti-PGLYRP1 mAbs that can block TREM-1responses, wtbalb/c mice were immunized with recombinant human PGLYRP1.Primary screening was done by means of direct ELISA on PGLYRP1 protein,and all PGLYRP1-specific hybridoma supernatants were subsequently testedin the BWZ/hTREM-1 reporter cell assay to identify monoclonalanti-PGLYRP1 antibodies capable of inhibiting TREM-1 activation inducedby PGN-stimulated neutrophils, as described under example 1. Thebioassay was run as follows: 40,000 hTREM-1/BWZ.36 cells/well wereplated in a clear bottom, black 96 well plate in the presence of 75ng/ml PGLYRP1 (SEQ ID NO: 1) with 2.5 μg/ml PGN-ECndi (Cat. no,tlrl-kipgn, Invivogen San Diego, Calif., USA) to provide a sub-maximalpositive signal, or alternatively in the presence of a sub-maximal level(1 μg/ml) of plastic adsorbed anti TREM-1 monoclonal antibody (Cat. no.MAB1278 R&D Systems, Minneapolis, Minn., USA) to provide a positivesignal. Test antibodies were titered into the assay starting at 10μg/ml, with 5 serial 2-fold dilutions. The assay was incubated overnightat 37 C, and developed with Beta Glo (Cat. no. E4740,Promega Madison,Wis., USA), as per the Beta Glo protocol, and luminescence was recorded.Data was plotted showing Beta Glo relative luminescent units vs testantibody concentration. Non-neutralizing negative control mlgG1 (Cat.no. MAB002, R&D Systems Minneapolis, Minn., USA) and neutralizingpositive control polyclonal goat anti hPGLYRP1 antibody (Cat. no.AF2590, R&D Systems, Minneapolis, Minn., USA) were run on each assayplate. As shown in FIG. 6A, F10, F14, F29, F36, F38, F77, F95 and F105were identified as being able to block the TREM-1 response to PGLYRP1.FIG. 6B shows other PGLYRP1 antibody hybridoma supernatants able toblock the TREM-1 dependent signal. M-hPGRPS-2F5 (SEQ ID NOs: 31-32 and-2F7 (SEQ ID NOs: 35-36) are very efficient blockers. In contrast,testing of commercially available monoclonal anti-PGLYRP1 antibodies inthe same assay protocol alongside the antiPGLYRP1 goat polyclonalpositive control (Cat, no. AF2590, R&D Systems Minneapolis, Minn., USA)showed that none of these could block TREM-1 activation (FIG. 6C). Theanti PGLYRP1 antibodies tested include: 188C424 from Thermo Scientific(6D), 4H230 and 9A319 Mabs from US Biological (FIG. 6D, 6E); all failedto block PGLYRP1 stimulation of the TREM-1 bioassay. Clone 6D653 fromSanta Cruz Biotechnology (FIG. 6F and 6G) had was found to block theassay non-specifically based on the observation that the Mab blockedboth PGLYRP1 (6F) and anti-TREM-1 stimulation (6G) while the positivecontrol anti PGLYRP1 polyclonal Ab only blocked PGLYRP1 mediatedactivation. This non-specific blocking may be due to azide toxicity onthe bioassay.

In conclusion, PGLYRP1 monoclonal antibodies have been identified thatnot only bind PGLYRP1 but also neutralize its TREM-1 signallingactivity. The method used to identify these molecules provides a uniqueadvantage over routine methods used to identify PGLYRP1 antibodies,evidenced by the failure of available commercial antibodies toneutralize PGLYRP1.

Example 13: PGLYRP1 Antibodies Block a TREM-1 Specific Signal

PGLYRP1 hybridoma clones were sequenced and recombinantly expressed as ahIgG4 antibody. Two of these mAb 0182 (from 1F36) (SEQ ID 15 and 16) andmAb 0184 (from 1F105) (SEQ ID 23 and 24) were retested in theBWZ/hTREM-1 reporter cell assay as described in example 13. Theseanti-PGLYRP1 antibodies block the TREM-1 response in the BWZ/hTREM-1reporter cell assay in a dose-dependent manner.

BWZ.36/hTREM-1 response, Beta Glo RLU Antibody Isotype 182 184 ug/ml AvgSD Avg SD Avg SD 0 74636 10004 74636 10004 74636 10004 0.16 70289 1301881858 3336 60738 5449 0.31 68555 5585 73382 650 59830 2837 0.63 6810511547 73831 7818 51198 397 1.25 71797 8545 63280 1663 46447 708 2.569207 5004 51675 1270 42062 1953 5 76951 901 33641 842 36194 1461 1083930 8962 20655 1080 25239 407 20 74555 511 11852 464 21333 115 4072296 8228 7696 306 15693 1861

This example shows that, in contrast to the commercial availableantibodies against PGLYRP1 shown in FIG. 6C-G, the PGLYRP1 antibodiesdisclosed herein are able to block the TREM-1 specific signal in theBWZ/hTREM-1 reporter cell assay.

Example 14: TNF-Alpha Release from M2 Macrophages Stimulated by PGLYRP1

Monocytes were differentiated into M2 macrophages and stimulated withPGLYRP1 complex. Antibodies (10 μg/ml) mAb −0182 and −0184 directedagainst PGLYRP1 is able to lower the TNF-alpha release. M2 macrophageswere differentiated as described in Example 10. Antibodies to be testedwere prepared at 4× concentrations in growth media and 50microliters/well were added. The final step in initiating the bioassaywas the addition of 100 microliters/well of M2 macrophage cells preparedas described above. PGLYRP1 monoclonal antibodies (mAb 0182 and mAb0184) were tested for neutralizing activity on M2 macrophages. Duplicate(unless otherwise noted) test wells were tested under the followingconditions: no added stimulation, 7.5 ng/ml PGLYRP1 only, 3 μg/ml PGN-BS(Cat. no. tlrl-pgnbs, Invivogen San Diego, Calif., USA) only(sextuplicates), PGLYRP1 with PGN-BS (sextuplicates), and PGLYRP1 withPGN-BS in the presence of PGLYRP1 antibodies or hIgG4 isotype controlantibody titrated in at concentrations between 40 μg/ml and 0.31 μg/mlin 2 fold dilutions.

Donor 1 Donor 2 TNF-α, pg/ml TNF-α, pg/ml M2 macrophages with: Avg SDAvg SD No addition 108 42 68 16 PGLYRP1 167 98 89 33 PGN 424 105 635 156PGLYRP1 + PGN 1660 198 2168 210 PGLYRP1 + PGN + isotype 1726 182 2483251 PGLYRP1 + PGN + mAb 0182 1322 173 2014 107 PGLYRP1 + PGN + mAb 01841207 168 1948 173

This example illustrates that the TREM-1 ligand PGLYRP1 is able tofurther increase TNFa release from M2 macrophages from two differentdonors and that the antibodies disclosed herein are able to decreasesuch TNFa release. Therefore, these PGLYRP1 antibodies are potentiallyuseful as TREM-1 antagonists.

Example 15: Survey of Binding Interactions Between TREM-1 and PGLYRP1Related Molecules Confirms Specificity

Having identified TREM-1 as being able to bind to PGLYRP1 and activateTREM-1 in the presence of PGN, we set out to determine whether or notTREM-1 could bind to the other PGLYRP family members. PGLYRP1 wasartificially anchored in the cell-membrane through addition, at theN-terminus, of an intracellular (IC) and transmembrane domain (TM)derived from the Type II receptor MDL-1. The latter constructs weredenoted Type II PGLYRP1. In Type II 1.0 (SEQ ID NO: 37), the chargedamino acid of the native MDL-1 receptor TM is maintained, and efficientexpression of this protein, is dependent on co-expression of DAP12. Inthe Type II 2.0 PGLYRP1 construct (SEQ ID NO: 38), the charged TMresidue has been substituted with a neutral amino acid (Lysine toLeucine), enabling the protein to be expressed independently of eg.DAP12, and an epitope tag of DYKDDDDK (SEQ ID NO: 39) was added. Fulllength hPGLYRP2 (SEQ ID NO: 40), hPGLYRP3 (SEQ ID NO: 41), and hPGLYRP4(SEQ ID NO: 42]), cDNAs were synthesised as membrane anchored proteinsusing N terminal fusion of the TypeII 2.0 MDL1 N terminus as utilizedfor PGLYRP1 above, cDNAs were subcloned into a modified pTT5 expressionplasmid vector (Zhang J et al. Protein Expression and Purification, Vol.65, Issue 1, May 2009, pp 77-8), and transfected as described in example8 into HEK293-6E cells alongside an empty vector negative control(Mock). Cells were assayed by flow cytometry for both surface andintracellular binding of the following probes: goat anti-human PGLYRP1(PGRPS) (Cat. no. AF2590, R&D Systems, Minneapolis, Minn., USA); Mabanti-human PGLYRP2 (PGRP-L) (Cat. no, MAB0755, Abnova, Walnut, Calif.,USA); Mab anti-human PGLYRP3 (PGRP-1a) (Cat. no. MAB0068, Abnova,Walnut, Calif., USA); Mab anti-human PGLYRP3 (PGRP-1a) (Cat. no.ab13901, Abcam, Cambridge Mass., USA); rabbit anti-human PGLYRP3(PGRP-1a) (Cat. no. 18082-1-AP, Protein Tech, Chicago Ill., USA); goatanti-human PGLYRP4-biotin (PGRP-1b) (Cat. no. BAF3018, R&D Systems,Minneapolis, Minn., USA); goat anti-mouse PGLYRP1 (PGRPS) (Cat. no.AF2696, R&D Systems, Minneapolis, Minn., USA); huTREM-1.Fc dimer(C0099), huTREML1.Fc dimer (CO246), huTREML2.Fc dimer (CO247);huTREM2,Fc dimer (C0248); The binding protocol was conducted as follows,using either cytofix/perm buffer (Cat. no. 51.2090KZ, BD Biosciences,San Jose Calif., USA) for intracellular staining or 2% FBS/PBS forsurface staining: cell pellets were resuspended in a 96-well roundbottom plate (to begin with: 160,000 cells/well) with 200 μlcytofix/perm buffer for 15 minutes at 22 C, washed twice with 200 μl 1×PermWash Buffer (diluted 10× in DiH20), stained with 50 μl probe dilutedto 5 □g/ml in 1× PermWash buffer, incubated for 1 hour at 4 C, cellswere then washed with 200 μl 1× PermWash Buffer, a secondary probe wasadded in 50 μl 1× PermWash buffer, cells were incubated at 4 C for 30minutes, washed twice with 200 μl 1× PermWash Buffer, pellets wereresuspended in 50 μl 1 1:1 PBS diluted CytoFix (BD:554655, San Jose,Calif.), incubated for 5 minutes at 22 C, 150 μl PBS/2% FBS was added,centrifuged for 5 minutes at 300 g, washed 1× with 200 μl PBS/2% FBS andresuspended in 100 μl PBS/2% FBS. Binding was analysed using FACS onLSRII (BD, San Jose Calif., USA).

As summarised in the table below, TREM-1 probe bound exclusively tohPGLYRP1 and no hTREM family member binding to PGLYRP2, PGLYRP3 orPGLYRP4 was detected.

Type II Type II Type II Type II Type II Type II Mock PGLYRP1 PGLYRP2PGLYRP3 PGLYRP4 PGLYRP3 + 4 muPGLYRP1 anti huPGLYRP1 − ++++ − − ++ +++++ (R&D, AF2590) anti huPGLYRP2 − − ++++ − − − n/d (Abnova, MAB07755)anti huPGLYRP3 − − − − − − n/d (Abnova, MAB0068) anti huPGLYRP3 − − − −− − n/d (Abcam, ab13901) anti huPGLYRP3 − − − − − − n/d (Proteintech,18082-1-AP) anti huPGLYRP4 − ++++ − − ++++ ++++ n/d (R&D, BAF3018) antimuPGLYRP1 − ++++ n/d n/d n/d n/d ++++ (R&D, AF2696) huTrem1.Fc (C0099) −++++ − − − − − huTremL1.Fc (C0246) − − − − − − − huTremL2.Fc (C0247) − −− − − − n/d huTrem2.Fc (C0248) − − − − − − n/d

Human membrane anchored PGLYRP1, PGLYRP2, PGLYRP3 and PGLYRP4 weretransiently expressed in HEK293 and probed with both in-house andcommercial soluble receptors and antibodies in order to identify newinteractions between family members. Binding scores were expressed as“n/d”, “−”, “+” or “++++”. Scores are a ratio of mean florescentintensity (MFI) of probe staining over negative control staining; “−”equals a ratio of <1, “++++” represents a score of >30 and “++”represents approximately 10-15, “+” represents 2-5 with a statisticallysignificant difference (p<0.05). Those skilled in the art maycharacterize this scoring as negative, bright or dim respectively.

TREM-1 only binds to PGLYRP1 and none of the other PGLYRP members, andvice versa: PGLYRP1 only interacts with TREM-1 and none of the otherTREM members.

Example 16: TREM-1 Ligand is Present in RA Synovial Fluid Samples

Rheumatoid arthritis is characterized by metacarpophalageal (MCP) jointinflammation in which activated granulocytes play a significant role.PGYLRP1 was assayed by ELISA and tested in BWZ/hTREM-1 reporter cellassay from synovial fluid drawn from MCP joints of 9 RA patients.Commercially sourced synovial fluid (Asterand, Detroit Mich., USA) wasthawed, vortexed and serially diluted in ELISA buffer and run in aPGLYRP1 assay following the manufacturers guidelines (Promega, MadisonWis., USA) Four of 9 patients showed elevated PGLYRP1 levels:

PGLYRP1, ng/ml RA SF donor Avg SD 1 14 5 2 8 1 3 213 44 4 229 58 5 13549 6 47 4 7 39 13 8 116 33 9 32 1

The RA synovial fluid samples were subsequently assayed for TREM ligandactivity in the BWZ reporter assay as described in example 2. Brieflysynovial fluid was thawed, vortexed, and serially diluted, assayed induplicate +/−10μg/ml PGNECndi (Invivogen, San Diego, Calif., USA) withthe addition of polyclonal PGLYRP1 antibody (Cat. no. AF2590, Promega,Madison Wis., USA) or a negative control polyclonal. Plastic adheredmonoclonal TREM-1 and isotype antibodies (R&D Systems, MinneapolisMinn., USA) served as positive and negative controls respectively. FIG.8 illustrates an example of how the synovial fluid from a rheumatoidarthritis patient is able to trigger the BWZ/hTREM-1 reporter cell assayin a PGLYRP1 dependent manner.

Example 17: Assembly of Mammalian Expression Vectors Useful in theIdentification and Characterization of the PGLYRP1: TREM 1 Interaction

A.) Construction of pJSV002 hTREM-1-G4S×3-hTREM-1/Fc6mut (SEQ ID NO: 9)

An Fc receptor binding deficient version of human IgG1 was built byeliminating the first 215 amino acids of human IgG1 comprising thevariable and constant one (CH1) domain and making the following aminoacid substitutions within the hinge, constant 2 and constant 3 domains:(E216G, C220S, L234A, L235E, G237A, A3305, P331S). This construct wasgiven the name Fc6mut since six mutations were made to modulate bindingto Fc receptors while a 7^(th) mutation (E216G) was incorporated tocreate a ApaI restriction cloning site. These mutations were built intopJSV002 (modified pTT5, Zhang J et al. Protein Expression andPurification, Vol, 65, Issue 1, May 2009, pp 77-8) allow cloning ofextracellular domains of receptors 5′ of Fc6mut as EcoRI/ApaI fragments.A cDNA was synthesised with a 5′ EcoRI restriction site, a GCCACC Kozaksequence the CD33 leader sequence followed by the extracellular domainof human TREM-1 (aa17-200) with a interspaced KpnI restriction site andglycine-glycine-glycine-serine spacer repeated three times (G4S×3)followed by an additional copy of the extracellular domain of humanTREM-1 (aa17-200) and an Apa1 site to allow cloning upstream of theFc6mut. This synthesised DNA was cut with EcoRI and ApaI and ligatedinto pJSV002 Fc6mut that had also been prepared with an EcoRI/ApaIdigestion. This ligation was electroporated into DH10B E. coli andplated onto ampicillin agar plates. Individual clones were grownovernight in 2 mls LB+ampicillin cultures and miniprepped followed byrestriction screening with EcoRI/ApaI to find clones with theappropriate 1219 base pair insert. Correct clones were sequenced, andone of the correct clones (#519) was selected for preparation ofadditional DNA by big prep.

B.) Construction of hTREM-1-COMP-SBP38x2-6His (SEQ ID NO: 10)

To create a pentameric TREM ECD molecule, a C terminal epitope tag wascreated in the pJSV002 expression vector. A synthetic cDNA encoding the3′ end of cartilage oligomeric protein (COMP) was fused to two copies ofthe streptavidin binding protein domain (SBP) followed by a C-terminal6xHis domain, built into pJSV002 such that extracellular domains ofreceptors could be cloned 5′ as of this fragment, as an EcoRI/KpnIfragment. Subsequently a hTREMcDNA was synthesised containing an EcoRIrestriction site followed by GCCACC Kozak sequence and the extracellulardomain of human TREM-1 with a C-terminal KpnI site. This EcoRI/KpnIfragment was ligated into pJSV002 COMP-SBP38x2-6His vector describedabove. This ligation was electroporated into DH10B E.coli (LifeTechnolohies, Carlsbad Calif., USA) and plated onto ampicillin agarplates. Individual clones were grown overnight in 2 mls LB+ampicillincultures and miniprepped followed by restriction screening withEcoRI/KpnI to find clones with the appropriate 616 bp insert. Correctclones were sequenced, and one of the correct clones #525 was selectedfor preparation of additional DNA by big prep. The full length cDNA islisted as SEQ ID NO: 10.

C.) Construction of pJSV002 hCD83-G4S×3-hCD83/Fc6mut (SEQ ID NO: 11)

hCD83 tetramer has previously been shown to have low binding by FACSanalysis when tested against a wide range of cell lines and thereforemade an excellent negative control for the IPMS experiment outlined inexample 3. To create this molecule, a cDNA was synthesised with a 5′EcoRI restriction site, a GCCACC Kozak sequence, the CD33 leadersequence followed by the extracellular domain of human CD83 with ainterspaced KpnI restriction site and glycine-glycine-glycine-serinespacer repeated three times (G4S×3) followed by an additional copy ofthe extracellular domain of human CD83. This cDNA was then clonedupstream of the hFc6mut in the pJSV002 expression vector previouslydescribed. The resulting mature protein is listed as SEQ ID NO: 6 andthe tandem CD83 extracellular domain cDNA sequence between EcoR1 andApa1 is shown as SEQ ID NO: 11.

D.) Construction of pJSV002 NCOMP-hDCIR (SEQ ID NO: 12)

As a negative control for the hTREM-1-COMP pentamer used in example 5,hDCIR-COMP was used. A pJSV002 based expression plasmid was created withthe following elements: 6xHIS tag followed by two copies of thestreptavidin binding protein domain (SBP) fused to the 3′ end ofcartilage oligomeric protein (COMP) such that extracellular domains oftype 2 receptors could be cloned 3′ of this fragment as an BgIII/BamHIfragments and expressed as pentameric soluble receptors. A PCR fragmentwas amplified from a synthetic cDNA template to generate a DNA fragmentwith BgIII and BamHI ends on the 5′ and 3′ ends respectively. Thisfragment was cut with BgIII and BamHI restriction enzymes followed byband purification. The resulting fragment was ligated into pJSV002 NCOMPthat had been previously cut with BgIII and BamHI. The ligation waselectroporated into DH10B E. coli and plated onto ampicillin selectionagar. Clones were picked, mini-prepped and screened with EcoRI and BamHIand clones with the proper 1.137 kB insert were sequenced. The cDNAcoding for the full open reading frame including NCOMP-SBP and DCIRsequence is designated SEQ ID NO: 12 and codes for the previouslyreferred to mature peptide sequence SEQ ID NO: 4.

E.) Construction of pNNC649-hTREM1-hFc6mut Dimer

TREM1-Fc dimer was used in Example 14 to confirm binding to PGLYRP1 andtest binding to other PGLYRP family members. A pTT5 based plasmid (ZhangJ et al., Protein Expression and Purification, Vol. 65, Issue 1, May2009, pp. 77-8) pNNC649 was utilized to allow cloning of extracellulardomains of receptors in frame and 5′ of Fc6mut. In order to expresshTREM1-Fc6mut, a cDNA was synthesised with a 5′ EcoRI restriction site,a GCCACC Kozak sequence and a hCD33 leader sequence followed by theextracellular domain of human TREM-1 (aa17-200) followed by a Kpn1 site.This cDNA was cloned into pNNC549 using restriction enzyme and DNAligase techniques familiar to those skilled in the art. The cDNA codingfor the full open reading frame including CD33 leader, hTREM1 ECD andFc6mut sequence is designated SEQ ID NO: 43 and codes for the maturepeptide sequence SEQ ID NO: 44.

F.) Construction of pNNC649-hTREML1-Fc6mut Dimer

A synthetic cDNA was created with a 5′ EcoRI restriction site, a GCCACCKozak sequence and a hCD33 leader sequence followed by the extracellulardomain of human TREML1 (aa16-162) followed by a KpnI site. This cDNA wascloned into pNNC549 vector previously described using restriction enzymeand DNA ligase techniques familiar to those skilled in the art. The cDNAcoding for the full open reading frame including CD33 leader, hTREML1ECD and Fc6mut sequence is designated SEQ ID NO: 45 and codes for themature peptide sequence SEQ ID NO: 46.

G.) Construction of pNNC649-hTREML2-Fc6mut Dimer

A synthetic cDNA was created with a 5′ EcoRI restriction site, a GCCACCKozak sequence and a hCD33 leader sequence followed by the extracellulardomain of human TREML2 (aa19-268) followed by a KpnI site. This cDNA wascloned into pNNC549 vector previously described using restriction enzymeand DNA ligase techniques familiar to those skilled in the art. The cDNAcoding for the full open reading frame including CD33 leader, hTREML2ECD and Fc6mut sequence is designated SEQ ID NO: 47 and codes for themature peptide sequence SEQ ID NO: 48.

H.) Construction of pNNC649-hTREM2-Fc6mut Dimer

A synthetic cDNA was created with a 5′ EcoRI restriction site, a GCCACCKozak sequence and a hCD33 leader sequence followed by the extracellulardomain of human TREM2 (aa19-174) followed by a KpnI site. This cDNA wascloned into pNNC549 vector previously described using restriction enzymeand DNA ligase techniques familiar to those skilled in the art. The cDNAcoding for the full open reading frame including CD33 leader, hTREM2 ECDand Fc6mut sequence is designated SEQ ID NO: 49 and codes for the maturepeptide sequence SEQ ID NO: 50.

Example 18: A Multimerised PGLYRP1 Activates TREM-1

A counter-structure or ligand for TREM-1 was identified through bindinganalysis and IP/MS proteomics. The identity of the ligand, PGLYRP1 (orPGRP-S) was subsequently validated through specific blockage.

Interestingly, while binding of the soluble PGLYRP1 can be demonstratedto the TREM-1, activation of the TREM-1 by PGLYRP1 requires theconcurrent presence of a scaffolding agent such as NeutrophilExtracellular Traps (NETs) or PGN.

To test if such alternative, and multimerised formats of PGLYRP1 mightbind and/or activate TREM-1, a cell-associated PGLYRP1 protein wasdesigned and expressed. Two conceptually distinct PGLYRP1 constructswere tested. In one, a GPI-anchoring sequence motif was added to theC-terminal end of PGLYRP1. In another, PGLYRP1 was artificially anchoredin the cell-membrane through addition, at the N-terminus, of anintracellular (IC) and transmembrane domain (TM) derived from the TypeII receptor MDL-1. The latter constructs were denoted Type II PGLYRP1.In Type II 1.0 (SEQ ID NO: 37), the charged amino acid of the nativeMDL-1 receptor TM is maintained, and efficient expression of thisprotein, is dependent on co-expression of DAP12. In the Type II 2.0PGLYRP1 construct (SEQ ID NO: 38), the charged TM residue has beensubstituted with a neutral amino acid (Lysine to Leucine), enabeling theprotein to be expressed independently of eg. DAP12. The cDNAs encodingthese constructs were transiently expressed in HEK293 6E cells, cellswere harvested on day two post-transfection and analyzed for theirability to stimulate the reporter cell line BWZ/hTREM1. Type II PGLYRP1transfectants were co-incubated with BWZ/hTREM1 reporter cells in theabsence of PGN. TREM1 activation was read out after 18 hours using theBetaGlo reagent (Cat. no. E4720, Promega, Madison Wis., USA). Thetransfectants expressing Type II PGLYRP1 induced activation of TREM-1 inthe absence of PGN, to a level 24-fold higher than seen with controlcells transfected with empty expression vector. In contrast, theGPI-anchored PGLYRP1, immobilized via the C-terminus of PGLYRP1, did notmediate any TREM-1 activation. Expression of the membrane-bound andimmobilized PGLYRP1 protein at the cell-surface was ascertained by flowcytometry using a polyclonal anti-PGLYRP1 antibody (AF2590). Bothproteins, Type II PGLYRP1 and GPI-PGLYRP1, were demonstrated to indeedbe cell-surface expressed.

Construct Fold activity C-GPI PGLYRP1 2.0 ± 0.4 (n = 3) Type II 1.0PGLYRP1  24 ± 3.3 (n = 3)

The table above shows that PGLYRP1 bound to the cell membrane surfacevia a C-terminal GPI anchor is not as potent in inducing TREM-1 activityas a Type II PGLYRP1 protein bound to the cell membrane via theN-terminal part. This illustrates the importance of a free C-terminalPGLYRP1 part to be able to stimulate TREM-1.

Type II PGLYRP1 activation was further demonstrated to be inhibitedspecifically by anti-PGLYRP1 antibody. Addition of the polyclonal (Cat.no. AF2590, R&D Systems, Minneapolis Minn., USA) PGLYRP1 antibody athigh concentration (1 μg/100 μl assay volume) was thus able to totallyinhibit this PGLYRP1 induced activity.

Condition Activity Type II 1.0 PGLYRP1 100%  Type II 1.0 PGLYRP1 + IsoAb 98% Type II 1.0 PGLYRP1 + AF2590 9.7% 

Sequences at the very C-terminal domain of PGLYRP1 appear critical tothe ability to activate the TREM-1 receptor. Several constructs, whichshare as a common feature, the modification at the extreme C-terminus ofPGLYRP1, have thus been seen to not mediate activation of TREM1/BWZreporter activity, while the corresponding constructs, which in contrasthave been modified at the N-terminus, do exhibit activity.

Construct Activity PGLYRP1 Native +++ PGLYRP1 N-Flag +++ PGLYRP1 C-Flag− PGLYRP1 N-Fc +++ PGLYRP1 C-Fc (+) Type II 1.0 PGLYRP1 +++ PGLYRP1C-GPI (+)

This indicates the importance of a free C-terminal PGLYRP1 part to beable to stimulate TREM-1.

Interpretation and Biological Perspectives

The ability to activate the TREM-1 receptor using either the nativePGLYRP1-ligand in the presence of PGN or alternatively, novel PGLYRP1variants which have been demonstrated to overcome the need for PGN,clearly demonstrates that PGN is not an absolute co-factor requirementfor TREM-1 activation. The common feature of the various molecularPGLYRP1 formats which were demonstrated to confer PGN-independent TREM-1activation appear to be a high density format leading to the hypothesisthat the predominant role of PGN, when acting as co-factor for thenative ligand, is to provide a scaffold for multimerisation. In vivo,such a scaffold could be provided by Neutrophil Extracellular Traps(NETS) (Blood 2005, 106: 2551-58) or other naturally occurring matrixstructures, such as hyaluronic acid, proteoglycan structures, such asversican, aggrecan, decorin, or fibrin, all of which may multimerise orotherwise present PGLYRP1.

These findings suggest that modification of the C-terminal part ofPGLYRP1 reduces TREM-1 activation which in turn indicates that blockingthe C-terminal part of PGLYRP1 with an agent, such as an antibodydirected against the C-terminal part of PGLYRP1, would decrease theTREM-1 interaction and thereby TREM-1 stimulation.

Example 19: Type II PGLYRP1 is able to Induce TNFalpha Release inSynovial Tissue Cells from Rheumatoid Arthritis Patients

Synovial tissue samples were obtained from RA patients during total kneereplacement. Single suspension of synovial tissue cells was isolated bya digestion via 4 mg/ml of collagenase (Cat, no. 11088793001, Roche,Mannheim, Germany) and 0.1 mg/ml of DNase (Cat. no. 11284932001, Roche,Mannheim, Germany) for 1 h at 37 degree. The synovial tissue cells(1×10̂5/well in culture medium RPMI (Cat. no. 22400105, LifeTechnologies, Carlsbad Calif., USA)+10% FCS (Cat. no. S0115, BioChromAG, Berlin, Germany) were co-cultured with various doses of HEK cellstransiently transfected with type II PGLYRP1 under hypoxic condition.After 24 h incubation, cell supernatants were harvested, and cytokineswere measured by TNFa ELISA (Cat. no, DY210, R&D Systems, Minneapolis,Minn., USA).

Type II PGLYRP1 (HEK transfected)/ TNF-α (pg/ml) release Control HEK 1 ×10{circumflex over ( )}5 3 × 10{circumflex over ( )}4 1 × 10{circumflexover ( )}4 3 × 10{circumflex over ( )}3 1 × 10{circumflex over ( )}3 0IgG4 + Type II 121.17 114.08 95.02 54.56 57.87 33.47 IgG4 + Control55.65 63.73 57.99 33.78 36.40 36.32

This example shows that the TREM-1 ligand can induce TNF-alpha in adose-dependent manner in synovial tissue cells from rheumatoid arthritispatients.

Example 20: PGLYRP1 Antibodies Block the TREM-1 Mediated Signal inNeutrophils and Decrease IL-8 Release

Having demonstrated that neutrophils can release PGLYRP1 and thatneutrophils also express the TREM-1 receptor, we tested whetherneutrophil-derived PGLYRP1 can stimulate neutrophils in an autocrinemanner. Isolated neutrophils were stimulated with PGN-SA (Cat. no.tlrl-pgnsa, Invivogen, San Diego Calif., USA), and the release of IL-8into the culture medium was measured. A PGLYRP1 antibody, mAb 0184, wasable to decrease the PGN-SA-induced IL-8 release. Neutrophils wereisolated from human healthy donor whole blood as described in example 3and resuspended in RPMI/10% FBS. The cells were plated out at 1.5×10E6cells/ml, and triplicate test wells were tested under the followingconditions: no added stimulation, 10 μg/ml PGN-SA only, or 10 μg/mlPGN-SA in the presence of PGLYRP1 antibody or hIgG4 isotype controlantibody at 4 μg/ml. The samples were cultured 24 hours in a 37° C., 5%CO₂ incubator. Supernatants were then harvested and analysed for IL-8using the Bioplex Pro Human Cytokine IL-8 set (Cat. no. 171-B5008M,BioRad, Hercules Calif., USA).

IL-8, pg/ml Neutrophils stimulated with Avg SD No addition 52 3 PGN-SA1158 341 PGN-SA + isotype control 1195 144 PGN-SA + mAb 0184 449 50

This example illustrates that IL-8 release from neutrophils induced bystimulation with the bacterially derived PGN-SA can be reduced byanti-PGLYRP1 antibody. The TREM-1 ligand PGLYRP1 is thus an autocrinestimulant of neutrophils, and the PGLYRP1 antibody disclosed herein arepotentially useful in down-regulating neutrophil responses.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now be apparent to those of ordinary skill in the art.It is, therefore, to be understood that the appended embodiments areintended to cover all such modifications and changes as fall within thetrue spirit of the invention.

1-6. (canceled)
 7. A method of treating an autoimmune disease or chronicinflammation in a subject in need thereof, comprising administering tothe subject an antibody or antigen-binding fragment thereof thatspecifically binds to human Peptidoglycan Recognition Protein 1(PGLYRP1), wherein the antibody or antigen-binding fragment thereof iscapable of reducing, blocking, or interfering with the interaction ofPGLYRP1 with human Triggering Receptor Expressed on Myeloid cells-1(TREM-1).
 8. The method of claim 7, wherein the autoimmune disease isrheumatoid arthritis, psoriasis, psoriatic arthritis, Crohn's disease,ulcerative colitis, or systemic lupus erythematosus.
 9. The method ofclaim 7, wherein the antibody or antigen-binding fragment thereof is achimeric, humanized, or human antibody.
 10. The method of claim 7,wherein the antibody or antigen-binding fragment thereof has a K_(D) forthe interaction with PGLYRP1 of 1×10⁻⁹ M or less, as measured by surfaceplasmon resonance.
 11. The method of claim 7, wherein the antibody orantigen-binding fragment thereof is capable of reducing greater than 50%TREM-1 response in a BWZ/hTREM-1 reporter cell assay.
 12. The method ofclaim 7, wherein the antibody or antigen-binding fragment thereof iscapable of blocking greater than 50% of TREM-1 mediated IL-8 releasefrom isolated cultured neutrophils.
 13. The method of claim 7, whereinthe antibody or antigen-binding fragment thereof comprises a CDRH1sequence as set forth in amino acid residues 31 to 35 of SEQ ID NO: 15,a CDRH2 sequence as set forth in amino acids 50 to 66 of SEQ ID NO: 15,a CDRH3 sequence as set forth in amino acid residues 99 to 108 of SEQ IDNO: 15, a CDRL1 sequence as set forth in amino acid residues 24 to 34 ofSEQ ID NO: 16, a CDRL2 sequence as set forth in amino acid residues 51to 56 of SEQ ID NO: 16, and a CDRL3 sequence as set forth in amino acidresidues 89 to 97 of SEQ ID NO:
 16. 14. The method of claim 7, whereinthe antibody or antigen-binding fragment thereof comprises a CDRH1sequence as set forth in amino acid residues 31 to 35 of SEQ ID NO: 19,a CDRH2 sequence as set forth in amino acids 50 to 66 of SEQ ID NO: 19,a CDRH3 sequence as set forth in amino acid residues 99 to 109 of SEQ IDNO: 19, a CDRL1 sequence as set forth in amino acid residues 24 to 33 ofSEQ ID NO: 20, a CDRL2 sequence as set forth in amino acid residues 49to 55 of SEQ ID NO: 20, and a CDRL3 sequence as set forth in amino acidresidues 88 to 96 of SEQ ID NO:
 20. 15. The method of claim 7, whereinthe antibody or antigen-binding fragment thereof comprises a CDRH1sequence as set forth in amino acid residues 31 to 35 of SEQ ID NO: 23,a CDRH2 sequence as set forth in amino acids 50 to 66 of SEQ ID NO: 23,a CDRH3 sequence as set forth in amino acid residues 99 to 108 of SEQ IDNO: 23, a CDRL1 sequence as set forth in amino acid residues 24 to 33 ofSEQ ID NO: 24, a CDRL2 sequence as set forth in amino acid residues 49to 55 of SEQ ID NO: 24, and a CDRL3 sequence as set forth in amino acidresidues 88 to 96 of SEQ ID NO:
 24. 16. The method of claim 7, whereinthe antibody or antigen-binding fragment thereof comprises a CDRH1sequence as set forth in amino acid residues 31 to 35 of SEQ ID NO: 27,a CDRH2 sequence as set forth in amino acids 50 to 66 of SEQ ID NO: 27,a CDRH3 sequence as set forth in amino acid residues 99 to 106 of SEQ IDNO: 27, a CDRL1 sequence as set forth in amino acid residues 24 to 33 ofSEQ ID NO: 28, a CDRL2 sequence as set forth in amino acid residues 49to 54 of SEQ ID NO: 28, and a CDRL3 sequence as set forth in amino acidresidues 87 to 95 of SEQ ID NO:
 28. 17. The method of claim 7, whereinthe antibody or antigen-binding fragment thereof comprises a CDRH1sequence as set forth in amino acid residues 31 to 35 of SEQ ID NO: 31,a CDRH2 sequence as set forth in amino acids 50 to 66 of SEQ ID NO: 31,a CDRH3 sequence as set forth in amino acid residues 99 to 109 of SEQ IDNO: 31, a CDRL1 sequence as set forth in amino acid residues 24 to 35 ofSEQ ID NO: 32, a CDRL2 sequence as set forth in amino acid residues 51to 57 of SEQ ID NO: 32, and a CDRL3 sequence as set forth in amino acidresidues 90 to 98 of SEQ ID NO:
 32. 18. The method of claim 7, whereinthe antibody or antigen-binding fragment thereof comprises a CDRH1sequence as set forth in amino acid residues 31 to 35 of SEQ ID NO: 35,a CDRH2 sequence as set forth in amino acids 50 to 66 of SEQ ID NO: 35,and a CDRH3 sequence as set forth in amino acid residues 99 to 109, aCDRL1 sequence as set forth in amino acid residues 24 to 34 of SEQ IDNO: 36, a CDRL2 sequence as set forth in amino acid residues 50 to 56 ofSEQ ID NO: 36, and a CDRL3 sequence as set forth in amino acid residues89 to 96 of SEQ ID NO:
 36. 19. An antibody or antigen-binding fragmentthereof which is capable of specifically binding PGLYRP1 and reducingPGLYRP1-mediated TREM-1 activity.
 20. The antibody or antigen-bindingfragment thereof of claim 19, which is capable of competing withantibody 1F10, antibody 1F36 (mAb 0182), antibody 1F95, antibody 1F105(mAb 0184), antibody 2F5 or antibody 2F7 for binding to PGLYRP1 andreducing PGLYRP1-mediated TREM-1 activity.
 21. An isolatedpolynucleotide that encodes the antibody or antigen-binding fragmentthereof of claim
 19. 22. The isolated polynucleotide of claim 21,wherein: (i) the antibody or antigen-binding fragment thereof comprisesa variable heavy chain encoded by SEQ ID NO: 13 and a variable lightchain encoded by SEQ ID NO: 14; (ii) the antibody or antigen-bindingfragment thereof comprises a variable heavy chain encoded by SEQ ID NO:17 and a variable light chain encoded by SEQ ID NO: 18; (iii) theantibody or antigen-binding fragment thereof comprises a variable heavychain encoded by SEQ ID NO: 21 and a variable light chain encoded by SEQID NO: 22; (iv) the antibody or antigen-binding fragment thereofcomprises a variable heavy chain encoded by SEQ ID NO: 25 and a variablelight chain encoded by SEQ ID NO: 26; (v) the antibody orantigen-binding fragment thereof comprises a variable heavy chainencoded by SEQ ID NO: 29 and a variable light chain encoded by SEQ IDNO: 30; or (vi) the antibody or antigen-binding fragment thereofcomprises a variable heavy chain encoded by SEQ ID NO: 33 and a variablelight chain encoded by SEQ ID NO:
 34. 23. A vector comprising theisolated polynucleotide of claim
 21. 24. A cell comprising the isolatedpolynucleotide of claim
 21. 25. A method of making an antibody orantigen-binding fragment thereof which is capable of specificallybinding PGLYRP1 and reducing PGLYRP1-mediated TREM-1 activity,comprising expressing the antibody or antigen-binding fragment thereofin the cell of claim 24.